Formulations of guanylate cyclase c agonists and methods of use

ABSTRACT

The invention provides low-dose formulations of guanylate cyclase-C (“GCC”) agonist peptides and methods for their use. The formulations of the invention can be administered either alone or in combination with one or more additional therapeutic agents, preferably an inhibitor of cGMP-dependent phosphodiesterase or a laxative.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/845,644 filed Sep. 4, 2015, now U.S. Pat. No. 9,610,321, which is acontinuation of U.S. patent application Ser. No. 14/661,299, filed Mar.18, 2015, which is a continuation of U.S. patent application Ser. No.13/421,769, filed Mar. 15, 2012, which is a continuation-in-part ofPCT/US2011/051805 filed on Sep. 15, 2011, which claims the benefit ofpriority to U.S. Provisional Application No. 61/383,156 filed on Sep.15, 2010, U.S. Provisional Application No. 61/387,636 filed on Sep. 29,2010, and U.S. Provisional Application No. 61/392,186 filed on Oct. 12,2010, the contents of which are incorporated by reference in theirentireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named“SYPA_009_C04US_Sequence_Listing.txt”, which was created on Mar. 23,2017 and is 113 KB in size, are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to low-dose formulations of guanylatecyclase C peptide agonists useful for the treatment and prevention ofvarious diseases and disorders.

BACKGROUND OF THE INVENTION

Guanylate cyclase C is a transmembrane form of guanylate cyclase that isexpressed on various cells, including gastrointestinal epithelial cells(reviewed in Vaandrager 2002 Mol. Cell. Biochem. 230:73-83). It wasoriginally discovered as the intestinal receptor for the heat-stabletoxin (ST) peptides secreted by enteric bacteria and which causediarrhea. The ST peptides share a similar primary amino acid structurewith two peptides isolated from intestinal mucosa and urine, guanylinand uroguanylin (Currie, et al., Proc. Nat'l Acad. Sci. USA 89:947-951(1992); Hamra, et al., Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993);Forte, L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell 63:941-948(1990); Guba, et al., Gastroenterology 111:1558-1568 (1996); Joo, etal., Am. J. Physiol. 274:G633-G644 (1998)).

In the intestines, guanylin and uroguanylin act as regulators of fluidand electrolyte balance. In response to high oral salt intake, thesepeptides are released into the intestinal lumen where they bind toguanylate cyclase C localized on the luminal membrane of enterocytes(simple columnar epithelial cells of the small intestines and colon).The binding of the guanylin peptides to guanylate cyclase C induceselectrolyte and water excretion into the intestinal lumen via a complexintracellular signaling cascade that is initiated by an increase incyclic guanosine monophosphate (cGMP).

The cGMP-mediated signaling that is initiated by the guanylin peptidesis critical for the normal functioning of the gut. Any abnormality inthis process could lead to gastrointestinal disorders such as irritablebowel syndrome (IBS) and inflammatory bowel diseases. Inflammatory boweldisease is a general name given to a group of disorders that cause theintestines to become inflamed, characterized by red and swollen tissue.Examples include ulcerative colitis and Crohn's disease. Crohn's diseaseis a serious inflammatory disease that predominantly affects the ileumand colon, but can also occur in other sections of the gastrointestinaltract. Ulcerative colitis is exclusively an inflammatory disease of thecolon, the large intestine. Unlike Crohn's disease, in which all layersof the intestine are involved, and in which there can be normal healthybowel in between patches of diseased bowel, ulcerative colitis affectsonly the innermost lining (mucosa) of the colon in a continuous manner.Depending on which portion of the gastrointestinal tract is involved,Crohn's disease may be referred to as ileitis, regional enteritis,colitis, etc. Crohn's disease and ulcerative colitis differ from spasticcolon or irritable bowel syndrome, which are motility disorders of thegastrointestinal tract. Gastrointestinal inflammation can be a chroniccondition. It is estimated that as many as 1,000,000 Americans areafflicted with inflammatory bowel disease, with male and female patientsappearing to be equally affected. Most cases are diagnosed before age30, but the disease can occur in the sixth, seventh, and later decadesof life.

IBS and chronic idiopathic constipation are pathological conditions thatcan cause a great deal of intestinal discomfort and distress but unlikethe inflammatory bowel diseases, IBS does not cause the seriousinflammation or changes in bowel tissue and it is not thought toincrease the risk of colorectal cancer. In the past, inflammatory boweldisease, celiac disease and IBS were regarded as completely separatedisorders. Now, with the description of inflammation, albeit low-grade,in IBS, and of symptom overlap between IBS and celiac disease, thiscontention has come under question. Acute bacterial gastroenteritis isthe strongest risk factor identified to date for the subsequentdevelopment of postinfective irritable bowel syndrome. Clinical riskfactors include prolonged acute illness and the absence of vomiting. Agenetically determined susceptibility to inflammatory stimuli may alsobe a risk factor for irritable bowel syndrome. The underlyingpathophysiology indicates increased intestinal permeability andlow-grade inflammation, as well as altered motility and visceralsensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key modulatorof gut function and is known to play a major role in pathophysiology ofIBS. The activity of 5-HT is regulated by cGMP.

While the precise causes of IBS and inflammatory bowel diseases (IBD)are not known, a disruption in the process of continual renewal of thegastrointestinal mucosa may contribute to disease pathology in IBD andaggravate IBS. The renewal process of the gastrointestinal lining is anefficient and dynamic process involving the continual proliferation andreplenishment of unwanted damaged cells. Proliferation rates of cellslining the gastrointestinal mucosa are very high, second only to thehematopoietic system. Gastrointestinal homeostasis depends on both theproliferation and programmed cellular death (apoptosis) of epithelialcells lining the gut mucosa. Cells are continually lost from the villusinto the lumen of the gut and are replenished at a substantially equalrate by the proliferation of cells in the crypts, followed by theirupward movement to the villus. The rates of cell proliferation andapoptosis in the gut epithelium can be increased or decreased in avariety of circumstances, e.g., in response to physiological stimulisuch as aging, inflammatory signals, hormones, peptides, growth factors,chemicals and dietary habits. In addition, an enhanced proliferationrate is frequently associated with a reduction in turnover time and anexpansion of the proliferative zone. The proliferation index is muchhigher in pathological states such as ulcerative colitis and othergastrointestinal disorders. Intestinal hyperplasia is a major promoterof gastrointestinal inflammation. Apoptosis and cell proliferationtogether regulate cell number and determine the proliferation index.Reduced rates of apoptosis are often associated with abnormal growth,inflammation, and neoplastic transformation. Thus, both increasedproliferation and/or reduced cell death may increase the proliferationindex of intestinal tissue, which may in turn lead to gastrointestinalinflammatory diseases.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of gastrointestinal mucosa by maintaining thebalance between proliferation and apoptosis. For example, uroguanylinand guanylin peptides appear to promote apoptosis by controllingcellular ion flux. Given the prevalence of inflammatory conditions inWestern societies a need exists to improve the treatment options forinflammatory conditions, particularly of the gastrointestinal tract.

Peptide agonists of guanylate cyclase C agonists (“GCC agonists”) aredescribed in U.S. Pat. Nos. 7,041,786, 7,799,897, and U.S. PatentApplication Publication Nos. US2009/0048175, US 2010/0069306, US2010/0120694, US 2010/0093635, and US 2010/0221329. However, theformulation of peptides for pharmaceutical delivery presents a number ofspecial problems. For example, peptides are subject to structuralmodifications by a variety of degradation mechanisms resulting inproblems of chemical and physical instability of the formulation.

SUMMARY OF THE INVENTION

The present invention provides low-dose formulations of peptide agonistsof guanylate cyclase C (“GCC”) and methods for their use in thetreatment and prevention of human diseases and disorders, such as agastrointestinal motility disorder, irritable bowel syndrome, afunctional gastrointestinal disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, functional dyspepsia, nonulcerdyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction; Crohn's disease, ulcerative colitis, inflammatorybowel disease, colonic pseudo-obstruction, obesity, congestive heartfailure, and benign prostatic hyperplasia. In certain embodiments, theformulations are stabilized against chemical degradation of the peptide.The low-dose formulations of the invention have unexpected efficacy inhumans in a dosage range that was not predicted based on studies inprimates. The formulations of the invention are particularly useful forthe treatment or prevention of chronic idiopathic constipation. Incertain embodiments, the GCC agonists are analogs of uroguanylin andbacterial ST peptides. In preferred embodiments, the analogs havesuperior properties compared to the naturally occurring or “wild-type”peptides. Examples of such superior properties include a high resistanceto degradation at the N-terminus and C-terminus from carboxypeptidases,aminopeptidases, and/or by other proteolytic enzymes present in thestimulated human intestinal juices and human gastric juices. Examples ofGCC agonists that can be used in the formulations and methods of theinvention are described in more detail below and in U.S. Pat. Nos.7,041,786, 7,799,897, and U.S. Patent Application Publication Nos.US2009/0048175, US 2010/0069306, US 2010/0120694, US 2010/0093635, andUS 2010/0221329, each of which is incorporated herein by reference inits entirety.

The invention provides an oral dosage formulation comprising one or morepharmaceutically acceptable excipients and at least one GCC agonistpeptide, wherein the amount of GCC agonist peptide per unit dose is from0.01 mg to 10 mg, and wherein the GCC agonist peptide is selected fromthe group consisting of SEQ ID NOs: 1-54 and 56-249. In one embodiment,the GCC agonist peptide has a chromatographic purity of no less than90%, no less than 90.5%, no less than 91%, no less than 92%, no lessthan 93%, no less than 94%, no less than 95%, no less than 96%, no lessthan 97%, no less than 98%, or no less than 99%. The chromatographicpurity of the GCC agonist peptide is determined as area percent by HPLC.In one embodiment, the GCC agonist peptide is selected from the groupconsisting of SEQ ID NOs: 1, 8, 9, or 56. In one embodiment, the GCCagonist peptide is selected from the group consisting of SEQ ID NOs: 1and 9. In one embodiment, the GCC agonist peptide is selected from thegroup consisting of SEQ ID NOs: 8 and 9. In one embodiment, the amountof GCC agonist peptide per unit dose is 0.1 mg, 0.3 mg, 0.6 mg, 1.0 mg,3.0 mg, 6.0 mg, 9.0 mg or 9.5 mg.

In one embodiment, the GCC agonist peptide has a total impurity contentof no greater than 10%, no greater than 9.5%, no greater than 9%, nogreater than 8%, no greater than 7%, no greater than 6%, no greater than5%, no greater than 4%, no greater than 3%, no greater than 2%, or nogreater than 1%. The total impurity content is determined as total areapercentages of impurities by HPLC. The impurities do not include anypharmaceutically acceptable excipient used for the formulation. In oneembodiment, the formulation is substantially free of inorganic acids andcarboxylic acids, e.g., HCl, phosphoric acid, or acetic acid. In thiscontext, carboxylic acids do not include amino acids or peptides. Inthis context “substantially” free of acids means that the acid contentof the formulation at the time of packaging is preferably less than0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than0.005%, or less than 0.001% of the total weight of the formulation. Inone embodiment, the formulation is free of HCl.

In one embodiment, the formulation is a solid formulation. In oneembodiment, the formulation is in the form of a powder, granule, sachet,troche, tablet, or capsule. In another embodiment, the formulation is aliquid formulation and the GCC agonist peptide is in solution orsuspension in a lipophilic liquid. In one embodiment, the liquid is arefined specialty oil or a medium chain triglyceride or related ester.In one embodiment, the refined specialty oil is selected from Arachisoil, Castor oil, cottonseed oil, maize (corn) oil, olive oil, sesameoil, soybean oil, and sunflower oil. In one embodiment, the medium chaintriglyceride or related ester is AKOMED E, AKOMED R, CAPTEX 355,LABRAFAC CC, LABRAFAC PG, LAUROGLYCOL FCC, MIGLYOL 810, MIGLYOL 812,MIGLYOL 829, MIGLYOL 840, and SOFTISAN 645. In one embodiment, theliquid is selected from the group consisting of medium chaintriglycerides, propylene glycol dicaprylocaprate, vitamin E, soybeanoil, Cremaphor, PG, and PG 400. In one embodiment, the unit dose is apowder, tablet, or capsule. In one embodiment, the unit dose is aliquid-filled capsule. In one embodiment, the capsule or tablet is in ablister pack or strip. Preferably, the blister pack or strip is made ofa material that is impermeable to water vapor and oxygen. In oneembodiment the blister pack is comprised of a metal foil. In oneembodiment the blister pack is a FOIL/FOIL blister pack. In oneembodiment, the container of the blister pack is flushed with an inertgas such as nitrogen or argon. In one embodiment, the container furtherincludes a desiccant. In a preferred embodiment the desiccant is amolecular sieve. In one embodiment, the unit dose is in a high densitypolyethylene bottle having a seal. In one embodiment, the bottle furthercomprises a desiccant. In one embodiment, the bottle further comprisesan oxygen scavenger or molecular sieve. In one embodiment, the bottle isnearly impermeable to oxygen and water vapor (e.g., much moreimpermeable than a HDPE bottle), such as an OxyGuard bottle.

In one embodiment, the one or more pharmaceutically acceptableexcipients include an inert carrier. In one embodiment, the inertcarrier is a selected from mannitol, lactose, a microcrystallinecellulose, or starch. In one embodiment, the inert carrier has aparticle size of from 50 to 900 microns, from 50 to 800 microns, from 50to 300 microns, from 50 to 200 microns, from 75 to 150 microns, from 75to 200 microns, or from 75 to 300 microns.

In one embodiment, the GCC agonist peptide is stabilized againstchemical or physical degradation for a period of at least 18 months at30° C. and 65% relative humidity, or at least 18 months at 25° C. and60% relative humidity, or at least 18 months at 2-8° C.

In one embodiment, the one or more pharmaceutically acceptableexcipients include a divalent cation salt such as calcium chloride. Inone embodiment, the one or more pharmaceutically acceptable excipientscomprise an amino acid, such as leucine, histidine, or arginine, or anamine such TRIS or TRIS/HCl.

In one embodiment, the oral dosage formulation consists of the GCCagonist peptide described herein, an inert carrier (e.g., CelphereSCP-100, Avicel PH 102, or Avicel PH 112), and a lubricant (e.g.,magnesium stearate). In one embodiment, the formulation consists of theGCC agonist peptide, an inert carrier (e.g., Avicel PH 200), a divalentcation salt (e.g., calcium chloride or calcium ascorbate), an amino acid(e.g., leucine, histidine, or arginine) or a protective amine (e.g.,TRIS), a coating agent (e.g., Methocel ES Premium LV) and optionally alubricant (e.g., magnesium stearate) or another additive (e.g.,trehalose). In one embodiment, the formulation consists of the GCCagonist peptide, a binder (e.g., Provsolv SMCC 90 LM), and adisintegrant (e.g., Explotab). In one embodiment, the formulationconsists of the GCC agonist peptide, a diluent (e.g., Mannogem EZ), abinder (e.g., Provsolv SMCC 90 LM), a disintegrant (e.g., Explotab), alubricant (e.g., Pruv).

The invention also provides a process for making the oral dosageformulations described herein, wherein the process comprises a step ofdry granulation, wet granulation, or spray coating followed by drying.In another embodiment, the process comprises a step of dry mixing. In apreferred embodiment the step of dry mixing includes geometric blending.In one embodiment, the process comprises a step of direct compression.In one embodiment, the process for making the oral dosage formulationsdescribed herein is a spray coating-drying process which includes (a)providing an aqueous solution comprising: a GCC agonist peptide selectedfrom the group consisting of SEQ ID NOs: 1-54 and 56-249, and one ormore pharmaceutically acceptable excipients, wherein the concentrationof the GCC agonist peptide ranges from 10 to 60 mg/mL; and (b) applyingthe aqueous solution to a pharmaceutically acceptable carrier togenerate a GCC agonist peptide-coated carrier.

In one embodiment of the spray coating-drying process above, the one ormore pharmaceutically acceptable excipients comprise a divalent cationsalt wherein the divalent cation is selected from Ca²⁺, Mg²⁺, Zn²⁺, andMn²⁺. In one embodiment, the one or more pharmaceutically acceptableexcipients comprise an amino acid selected from leucine, isoleucine, andvaline. In one embodiment, the one or more pharmaceutically acceptableexcipients comprise a coating agent (such as hypromellose Methocel E5PremLV). In one embodiment, the aqueous solution has a pH greater than 4(e.g., 4.5-5.5, 5-6, about 5, or greater than 5) or even greater than 7.In one embodiment, the aqueous solution is substantially free ofinorganic acids and carboxylic acids. In one embodiment, the GCC agonistpeptide is selected from the group consisting of SEQ ID NOs: 1, 8, 9,and 56. In one embodiment, the process further includes drying the GCCagonist peptide-coated carrier.

The invention further provides an oral dosage formulation made by theprocess described herein. Preferably, the GCC agonist peptide as made isstabilized against chemical or physical degradation for a period of atleast 18 months at 30° C. and 65% relative humidity, or at least 18months at 25° C. and 60% relative humidity, or at least 18 months at2-8° C.

The invention also provides a method for treating or preventing adisease or disorder in a subject in need thereof, comprisingadministering to the subject an oral dosage formulation comprising atleast one GCC agonist peptide, wherein the amount of GCC agonist peptideper unit dose is from 0.01 mg to 10 mg, and wherein the GCC agonistpeptide is selected from the group consisting of SEQ ID NOs: 1-54 and56-249. Preferably, the subject is a human subject. In one embodiment,the GCC agonist peptide is selected from the group consisting of SEQ IDNOs: 1, 8, 9, or 56. In one embodiment, the GCC agonist peptide isselected from the group consisting of SEQ ID NOs: 1 and 9. In oneembodiment, the amount of GCC agonist peptide per unit dose is 0.1 mg,0.3 mg, 0.6 mg, 1.0 mg, 3.0 mg, 6.0 mg, 9.0 mg, 9.5 mg, or 10 mg.

In one embodiment, the disease or disorder is a gastrointestinal diseaseor disorder selected from the group consisting of irritable bowelsyndrome, non-ulcer dyspepsia, chronic intestinal pseudo-obstruction,functional dyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastro esophageal reflux disease, constipation, gastroparesis,heartburn, gastric cancer, and H. pylori infection. In a preferredembodiment, the gastrointestinal disease or disorder is chronicidiopathic constipation.

In one embodiment, the method further comprises administering to thesubject an effective amount of an inhibitor of a cGMP-specificphosphodiesterase. In one embodiment, the cGMP-dependentphosphodiesterase inhibitor is selected from the group consisting ofsuldinac sulfone, zaprinast, and motapizone, vardenifil, and suldenifil.

In one embodiment, the method further comprises administering to thesubject an effective amount of at least one laxative. In one embodiment,the at least one laxative is selected from the group consisting ofSENNA, MIRALAX, PEG, or calcium polycarbophil.

In one embodiment, the method further comprises administering to thesubject an effective amount of at least one anti-inflammatory agent.

The invention also provides pharmaceutical compositions comprising theformulations described herein.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Plecanatide (SP-304) treatment reduced time to first BMfollowing daily dose.

FIG. 2: Effect of daily treatment with plecanatide on spontaneous bowelmovements (SBM) in chronic constipation patients.

FIG. 3: Effect of daily treatment with plecanatide on completespontaneous bowel movements (CSBM) in chronic constipation patients.

FIG. 4: Effect of daily treatment with plecanatide on Bristol Stool FormScores (BSFS) in chronic constipation patients.

FIG. 5: Effect of daily treatment with plecanatide on straining scoresin chronic constipation patients

FIG. 6: Percentage of subjects reporting improvements in abdominaldiscomfort scores after 14-days of daily treatment with plecanatide.

DETAILED DESCRIPTION

The invention provides pharmaceutical formulations of peptide GCCagonists. It is intended that the formulations of the invention are“pharmaceutical” formulations, meaning that they are suitable forpharmaceutical use. Accordingly, the term “formulations” as used hereinis meant to encompass pharmaceutical formulations even if“pharmaceutical” is not expressly stated. Pharmaceutical compositionscomprising the formulations described herein are also provided by theinvention. The formulations of the invention preferably providestability against chemical and physical degradation of the peptide,e.g., plecanatide (i.e., SEQ ID #1).

The invention is based in part upon the discovery that mannitol mixesvery effectively with the GCC agonist peptides described herein andprovides stability against degradation, allowing the peptides to beformulated at very low doses. The invention is also based in part on thediscovery that very low doses of the GCC agonist peptides describedherein are effective for the treatment of diseases and disorders inhumans. The dosage range found to be effective was not predicted basedon animal studies. The invention is also based in part upon thediscovery that a divalent cation (e.g., Ca²⁺) and/or an amino acid(e.g., leucine or arginine) stabilize the GCC agonist peptides describedherein during a process (e.g., spray coating-drying process) ofmanufacturing a formulation of the GCC agonist peptides and providesstability against degradation both during the manufacturing process andstorage of the formulation.

Plecanatide is a charged peptide due to the presence of four carboxylicacids and single amine group with a calculated pKa of approximately 3.5.Therefore plecanatide is likely to interact with ions in solution or inthe solid state. Plecanatide is a hygroscopic peptide requiring thecontrol of water during manufacture and storage to promote long termstability. Plecanatide is prone to degradation by oxidation in thepresence of residual peroxides or formaldehyde contaminants that areformed from peroxide reaction with polymeric excipients. The presentinvention discloses a manufacturing process and dry solid formulationcompositions that minimizes water content. The formulations arecomprised of components to minimize levels of residual formaldehyde andperoxides commonly found in many pharmaceutical excipients. Theinvention also discloses additives (i.e. CaCl₂) that may function aslocal desiccants in the formulation. Divalent cation salts such ascalcium ascorbate, MgCl₂, ZnCl₂, MnCl₂ and CaCl₂ bind plecanatide andsterically hinder reactive species such as water or oxygen from causingplecanatide degradation by molecular displacement. The invention furtherincludes scavengers of residual formaldehyde (amines such as TRIS orTRIS/HCl or amino acids such as leucine, isoleucine and valine), anddiscloses packaging confirmations to minimize oxygen exposure and watervapor during storage. The invention also discloses a stablemanufacturing process comprised of initially dissolving plecanatide incold water to minimize solution degradation, followed by spray coatingthe peptide solution on particles and drying to remove moisture.

The formulations of the invention are particularly useful for thetreatment or prevention of a gastrointestinal disease or disorderselected from the group consisting of irritable bowel syndrome,non-ulcer dyspepsia, chronic intestinal pseudo-obstruction, functionaldyspepsia, colonic pseudo-obstruction, duodenogastric reflux, gastroesophageal reflux disease, chronic idiopathic constipation,gastroparesis, heartburn, gastric cancer, and H. pylori infection.

In one embodiment, the formulations of the invention are used in amethod for the treatment of constipation. Clinically accepted criteriathat define constipation range from the frequency of bowel movements,the consistency of feces and the ease of bowel movement. One commondefinition of constipation is less than three bowel movements per week.Other definitions include abnormally hard stools or defecation thatrequires excessive straining. Constipation may be idiopathic (functionalconstipation or slow transit constipation) or secondary to other causesincluding neurologic, metabolic or endocrine disorders. These disordersinclude diabetes mellitus, hypothyroidism, hyperthyroidism,hypocalcaemia, Multiple sclerosis, Parkinson's disease, spinal cordlesions, Neurofibromatosis, autonomic neuropathy, Chagas disease,Hirschsprung disease and cystic fibrosis. Constipation may also be theresult of surgery or due to the use of drugs such as analgesics (likeopioids), antihypertensives, anticonvulsants, antidepressants,antispasmodics and antipsychotics. In a preferred embodiment, theconstipation is chronic idiopathic constipation.

The stabilized formulations of the invention comprise at least one GCCagonist peptide formulated with one or more excipients such that thepeptide is stabilized against chemical degradation. Chemical degradationof peptides results from a number of mechanisms including oxidation,water-mediated degradation, and reaction with aldehydes or reducingsugars. The ideal excipient or combination of excipients will benon-hygroscopic, have few or no reducing sugars, and be substantiallyfree of contaminants such as iron, peroxide, and formaldehyde. Theformulations of the invention are preferably substantially free ofwater. In this context “substantially” free of water means that thewater content of the formulation at the time of packaging is preferablyless than 7%, less than 5%, less than 1%, or less than 0.5% of the totalweight of the formulation. In one embodiment the amount of water isbetween 0.1 to 5% of the total weight of the formulation. In oneembodiment, the amount of water in the formulation of the inventionmanuafactured through a spray-coating process is less than 0.5% (e.g.,about 0.47%).

In the context of the present formulations, the term “stable” or“stabilized” refers to the resistance of the peptide to chemical orphysical degradation over time. Preferably, a stable formulation of theinvention retains an amount of the peptide in the formulation over aperiod of time that is at least 90%, preferably at least 95%, and mostpreferably at least 99% the amount of peptide initially present in theformulation. In one embodiment, a stable formulation of the invention,over a period of time (e.g., 18 month), has an increase in the totalimpurity content not greater than 8%, not greater than 7%, not greaterthan 6%, not greater than 5%, not greater than 4%, not greater than 3%,not greater than 2%, or not greater than 1%. In one embodiment, thepeptide is chemically stable in the formulation for a period of timethat is at least 18 months, at least 20 months, or at least 24 monthswhen stored at 25 degrees Celsius (25 C) and 60% relative humidity. Inone embodiment, the peptide is chemically stable in the formulation fora period of time that is at least 18 months, at least 20 months, or atleast 24 months when stored at 2-8 degrees Celsius (2-8 C). In oneembodiment, the peptide is chemically stable in the formulation for aperiod of time that is at least 3 months, 12 months, 18 months andpreferably 24 months when stored at 25 degrees Celsius (25 C) and 60%relative humidity. In one embodiment, the peptide is chemically stablein the formulation for a period of time that is at least 3 months, 18months and preferably 24 months when stored at 30 degrees Celsius (30C).

The low-dose formulations of the invention comprise an amount of atleast one GCC agonist peptide per unit dose that is less than 10 mg. Itis especially advantageous to formulate oral compositions in unit dosageform for ease of administration and uniformity of dosage. The term “unitdosage form” as used herein refers to physically discrete units suitedas unitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved. In one embodiment, the unit dosage form is atablet or a capsule.

In one embodiment of the low-dose formulations of the invention, theamount of GCC agonist peptide per unit dose is from 0.01 mg to 10 mg. Inone embodiment, the amount of GCC agonist peptide per unit dose is 0.1mg, 0.3 mg, 0.6 mg, 1.0 mg, 3.0 mg, 6.0 mg, 9.0 mg, 9.5 mg, or 10 mg.

In one embodiment, the low-dose formulation contains a carrier that isnon-hygroscopic. In one embodiment, the carrier is selected frommannitol and maltose (e.g., ADVANTOSE 100).

In one embodiment, the carrier is cellulose, preferably microcrystallinecellulose (e.g., Avicel PH 102, low moisture Avicel PH 112, Avicel PH200, or Celphere SCP-100). In one embodiment, the carrier is calciumphosphate or calcium sulphate. In another embodiment, the carrier is asaccharide. The term “saccharide” as used herein also refers topolysaccharides. Thus, the term saccharide is meant to includepolysaccharides. In one embodiment, the saccharide is selected frommannitol, trehalose, lactose, sucrose, sorbitol, and maltose. In apreferred embodiment, the saccharide is mannitol. Preferably thesaccharide has a low water content, a small particle size and a narrowparticle-size distribution.

Carriers having small particle sizes, and/or spherical shape, and narrowsize distribution are preferred. Particles of less than 20 microns havea relatively high surface area to volume ratio causing inter-particleattractive forces to dominate and resist bulk flow. Larger particles(greater than 100 microns) tend to roll or slide over one another andexhibit superior bulk flow properties compared with small particles. Anarrow particle-size distribution reduces particle packing and increasesflow. In one embodiment, the particles are between 20 and 500 microns insize (as measured across the largest diameter of the particle, onaverage). In one embodiment, a small particle size and a narrow particlesize range refers to particles having a size range of from 20-300microns, 50-200 microns, or 75-150 microns. In certain embodiments, thecarrier has a substantially spherical shape such as can be obtained witha spray drying process.

In one embodiment, the low-dose formulation is a solid formulation andthe unit dose is in the form of a tablet or capsule. In one embodiment,the low-dose formulation is a liquid formulation and the unit dosageform is a liquid-filled capsule. In one embodiment, the liquidformulation in the form of a solution or suspension of the GCC agonistpeptide in an lipophilic liquid. Examples of suitable liquids includemedium chain triglycerides (e.g., LABRAFAC Lipophile), propylene glycoldicaprylocaprate (e.g., LABRAFAC PG), vitamin E (e.g., a tocopherol),PEG 400 (e.g., Polyethylene glycol low M.W. (liquid)), propylene glycol,soybean oil, and Castor oil. In one embodiment, the liquid is selectedfrom the group consisting of medium chain triglycerides, propyleneglycol dicaprylocaprate, vitamin E, and soybean oil. In one embodiment,the refined specialty oil is selected from Arachis oil, Castor oil,cottonseed oil, maize (corn) oil, olive oil, sesame oil, soybean oil,and sunflower oil. In one embodiment, the medium chain triglyceride orrelated ester is AKOMED E, AKOMED R, CAPTEX 355, LABRAFAC CC, LABRAFACPG, LAUROGLYCOL FCC, MIGLYOL 810, MIGLYOL 812, MIGLYOL 829, MIGLYOL 840,and SOFTISAN 645.

A formulation according to the invention may be contained in a blisterpack. In a particular embodiment, the powder, tablet, or capsulecomprising the formulation is contained in a blister pack. Preferably,the blister pack is made of a material that allows only minimalpermeation by water vapor and oxygen. In one embodiment the blister packis comprised of a metal foil. In one embodiment, the blister pack iscomprised of ACLAR. In one embodiment, the container of the blister packis flushed with an inert gas such as nitrogen or argon. In oneembodiment, the container further includes a desiccant. In oneembodiment, the desiccant is calcium chloride. In one embodiment thedesiccant is a molecular sieve.

While any GCC agonist known in the art can be formulated according tothe present invention, analogs of uroguanylin and bacterial ST peptidesare preferred. In certain embodiments, the uroguanylin and bacterial STpeptide analogs have superior properties compared to naturallyoccurring, or “wild-type” peptides. For example, the uroguanylin andbacterial ST peptides for use in the present invention are preferablymodified to increase their resistance to degradation at the N-terminusand C-terminus from carboxypeptidases, aminopeptidases, and/or by otherproteolytic enzymes present in the stimulated human intestinal juicesand human gastric juices. In certain embodiments, the GCC agonistformulation comprises a peptide consisting essentially of an amino acidsequence selected from SEQ ID NOs: 1-249. In a preferred embodiment, thepeptide consists essentially of an amino acid sequence selected from SEQID NOs: 1, 8, 9, 55 and 56. The term “consists essentially of” refers toa peptide that is identical to the reference peptide in its amino acidsequence or to a peptide that does not differ substantially in terms ofeither structure or function from the reference peptide. A peptidediffers substantially from the reference peptide if its primary aminoacid sequence varies by more than three amino acids from the referencepeptide or if its activation of cellular cGMP production is reduced bymore than 50% compared to the reference peptide. Preferably,substantially similar peptides differ by no more than two amino acidsand not by more than about 25% with respect to activating cGMPproduction. In preferred embodiments, the GCC agonist is a peptidecomprising at least 12 amino acid residues, and most preferablycomprising between 12 and 26 amino acids. Non-limiting examples of suchanalogs of uroguanylin and bacterial ST peptides are described inSection 1.2 below.

The invention provides methods for treating or preventing certaindiseases and disorders and methods for increasing gastrointestinalmotility in a subject in need thereof by administering an effectiveamount of a GCC agonist formulation to the subject. The term “treating”as used herein refers to a reduction, a partial improvement,amelioration, or a mitigation of at least one clinical symptomassociated with the gastrointestinal disorders being treated. The term“preventing” refers to an inhibition or delay in the onset orprogression of at least one clinical symptom associated with thegastrointestinal disorders to be prevented. The term “effective amount”as used herein refers to an amount that provides some improvement orbenefit to the subject. In certain embodiments, an effective amount isan amount that provides some alleviation, mitigation, and/or decrease inat least one clinical symptom of the gastrointestinal disorder to betreated. In other embodiments, the effective amount is the amount thatprovides some inhibition or delay in the onset or progression of atleast one clinical symptom associated with the gastrointestinal disorderto be prevented. The therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. The term“subject” preferably refers to a human subject but may also refer to anon-human primate or other mammal preferably selected from among amouse, a rat, a dog, a cat, a cow, a horse, or a pig.

In accordance with the methods of the present invention, the GCC agonistformulation can be administered alone or in combination with one or moreadditional therapeutic agents to prevent or treat inflammation, cancerand other disorders, particularly of the gastrointestinal tract. In apreferred embodiment, the GCC agonist formulation is administered forthe treatment of chronic constipation. In one embodiment, the GCCagonist formulation is administered in combination with one or moreadditional therapeutic agents selected from the group consisting ofphosphodiesterase inhibitors, cyclic nucleotides (such as cGMP andcAMP), a laxative (such as SENNA, METAMUCIL, MIRALAX, PEG, or calciumpolycarbophil), a stool softener, an anti-tumor necrosis factor alphatherapy for IBD (such as REMICADE, ENBREL, or HUMAIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters.

1.1 Formulations

The formulations of the invention contain one or more GCC agonistpeptides described herein, in combination with one or morepharmaceutically acceptable carriers (also referred to as diluents)and/or excipients. In a preferred embodiment, the formulations of theinvention include an inert carrier. The inert carrier is preferablynon-hygroscopic. In one embodiment, the carrier in the formulationcontains few or no reducing sugars and is substantially free ofcontaminants including, but not limited to, iron, peroxide, andformaldehyde. In one embodiment, the carrier is selected from the groupconsisting of sorbitol, mannitol, EMDEX, and starch. In one embodiment,the carrier is mannitol (e.g., MANNOGEM) or microcrystalline cellulose(e.g. PROSOLV, CELPHERE, CELPHERE beads).

The low-dose formulations of the invention contain no greater than 10 mgper unit dose of a GCC agonist peptide. The remainder of the formulationis comprised of the carrier and one or more optional excipients. In oneembodiment, the amount of carrier is at least 90% of the total weight ofthe formulation. In another embodiment, the amount of carrier is atleast 95% or at least 98% of the total weight of the formulation. In oneembodiment, the amount of carrier is between 90 and 99.9% of the totalweight of the formulation. In one embodiment, the one or more optionalexcipients comprise a disintegrant which is present at 1 to 5% of thetotal weight of the formulation. In one embodiment, the one or moreoptional excipients comprise a lubricant which is present at 0.02 to 5%of the total weight of the formulation. In one embodiment, the one ormore optional excipients comprise an amino acid such as arginine,leucine, isoleucine, valine, histidine, phenylalanine, alanine, glutamicacid, aspartic acid, glutamine, methionine, asparagine, tyrosine,threonine, tryptophan, or glycine, which is present at 0.1 to 4% (e.g.,0.1-1%) of the total weight of the formulation. In one embodiment, themolar ratio between the amino acid and the GCC agonist peptide is fromabout 2:1 to about 30:1 or about 2:1 to about 20:1 (e.g., 5:1). In oneembodiment, the one or more optional excipients comprise a stabilizersuch as a divalent cation salt, more specifically, a water-solubledivalent cation salt (e.g., calcium chloride, magnesium chloride, zincchloride, manganese chloride, or calcium ascorbate), which is present at0.1 to 12% (e.g., 0.1-4%) of the total weight of the formulation. In oneembodiment, the molar ratio between the salt and the GCC agonist peptideis from about 5:1 to about 20:1 (e.g., 10:1).

The formulations may contain other additives as needed, including forexample lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, raffnose, maltitol, melezitose, stachyose, lactitol,palatinite, starch, xylitol, mannitol, myoinositol, and the like, andhydrates thereof, and amino acids, for example alanine, glycine andbetaine, and polypeptides and proteins, for example albumen.

Further examples of pharmaceutically acceptable carriers and excipientsinclude, but are not limited to binders, fillers, disintegrants,lubricants, anti-microbial agents, antioxidant, and coating agents suchas: BINDERS: corn starch, potato starch, other starches, gelatin,natural and synthetic gums such as acacia, xanthan, sodium alginate,alginic acid, other alginates, powdered tragacanth, guar gum, celluloseand its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone (e.g., povidone, crospovidone, copovidone, etc),methyl cellulose, Methocel, pre-gelatinized starch (e.g., STARCH 1500®and STARCH 1500 LM®, sold by Colorcon, Ltd.), hydroxypropyl methylcellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook,Pa., USA), Emdex, Plasdone, or mixtures thereof, FILLERS: talc, calciumcarbonate (e.g., granules or powder), dibasic calcium phosphate,tribasic calcium phosphate, calcium sulfate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,dextrose, fructose, honey, lactose anhydrate, lactose monohydrate,lactose and aspartame, lactose and cellulose, lactose andmicrocrystalline cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose &amp; guar gum, molasses, sucrose, ormixtures thereof, DISINTEGRANTS: agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate (such asExplotab), potato or tapioca starch, other starches, pre-gelatinizedstarch, clays, other algins, other celluloses, gums (like gellan),low-substituted hydroxypropyl cellulose, ployplasdone, or mixturesthereof, LUBRICANTS: calcium stearate, magnesium stearate, mineral oil,light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol,other glycols, compritol, stearic acid, sodium lauryl sulfate, sodiumstearyl fumarate (such as Pruv), vegetable based fatty acids lubricant,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zincstearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL200, W.R. Grace Co., Baltimore, Md. USA), a coagulated aerosol ofsynthetic silica (Deaussa Co., Piano, Tex. USA), a pyrogenic silicondioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA), or mixtures thereof,ANTI-CAKING AGENTS: calcium silicate, magnesium silicate, silicondioxide, colloidal silicon dioxide, talc, or mixtures thereof,ANTIMICROBIAL AGENTS: benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride,cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben,phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate,phenylmercuric nitrate, potassium sorbate, propylparaben, sodiumbenzoate, sodium dehydroacetate, sodium propionate, sorbic acid,thimersol, thymo, or mixtures thereof, ANTOXIDANTS: ascorbic acid, BHA,BHT, EDTA, or mixture thereof, and COATING AGENTS: sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin,pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropylmethylcellulose (hypromellose), hydroxypropyl methyl cellulosephthalate, methylcellulose, polyethylene glycol, polyvinyl acetatephthalate, shellac, sucrose, titanium dioxide, carnauba wax,microcrystalline wax, gellan gum, maltodextrin, methacrylates,microcrystalline cellulose and carrageenan or mixtures thereof.

The formulation can also include other excipients and categories thereofincluding but not limited to Pluronic®, Poloxamers (such as Lutrol® andPoloxamer 188), ascorbic acid, glutathione, protease inhibitors (e.g.soybean trypsin inhibitor, organic acids), pH lowering agents, creamsand lotions (like maltodextrin and carrageenans); materials for chewabletablets (like dextrose, fructose, lactose monohydrate, lactose andaspartame, lactose and cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose and guar gum, sorbitol crystalline);parenterals (like mannitol and povidone); plasticizers (like dibutylsebacate, plasticizers for coatings, polyvinylacetate phthalate); powderlubricants (like glyceryl behenate); soft gelatin capsules (likesorbitol special solution); spheres for coating (like sugar spheres);spheronization agents (like glyceryl behenate and microcrystallinecellulose); suspending/gelling agents (like carrageenan, gellan gum,mannitol, microcrystalline cellulose, povidone, sodium starch glycolate,xanthan gum); sweeteners (like aspartame, aspartame and lactose,dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses,sorbitol crystalline, sorbitol special solution, sucrose); wetgranulation agents (like calcium carbonate, lactose anhydrous, lactosemonohydrate, maltodextrin, mannitol, microcrystalline cellulose,povidone, starch), caramel, carboxymethylcellulose sodium, cherry creamflavor and cherry flavor, citric acid anhydrous, citric acid,confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake,disodium edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake,FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminumlake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 AluminumLake, FD&C Yellow No. 6, FD&C Yellow No. 10, glycerol palmitostearate,glyceryl monostearate, indigo carmine, lecithin, manitol, methyl andpropyl parabens, mono ammonium glycyrrhizinate, natural and artificialorange flavor, pharmaceutical glaze, poloxamer 188, Polydextrose,polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch,pregelatinized starch, red iron oxide, saccharin sodium, sodiumcarboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate,strawberry flavor, synthetic black iron oxide, synthetic red iron oxide,titanium dioxide, and white wax.

Solid oral dosage forms may optionally be treated with coating systems(e.g. Opadry® fx film coating system, for example Opadry® blue(OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white (YS-1-7040), andblack ink (S-1-8 106).

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(ε-caprolactone) and poly(alkylene oxide) (U.S.20030068384) to create a sustained release formulation. Other sustainedrelease formulations and polymers for use in the compositions andmethods of the invention are described in EP 0 467 389 A2, WO 93/24150,U.S. Pat. No. 5,612,052, WO 97/40085, WO 03/075887, WO 01/01964A2, U.S.Pat. No. 5,922,356, WO 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat.No. 5,968,895, U.S. Pat. No. 6,180,608, U.S. 20030171296, U.S.20020176841, U.S. Pat. No. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat.No. 5,134,122, U.S. Pat. No. 5,192,741, U.S. Pat. No. 5,192,741, U.S.Pat. No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. Pat. No. 5,445,832U.S. Pat. No. 4,931,279, U.S. Pat. No. 5,980,945, WO 02/058672, WO97/26015, WO 97/04744, and US20020019446. In such sustained releaseformulations microparticles (Delie and Blanco-Prieto 2005 Molecule10:65-80) of polypeptide are combined with microparticles of polymer.U.S. Pat. No. 6,011,01 and WO 94/06452 describe a sustained releaseformulation providing either polyethylene glycols (i.e. PEG 300 and PEG400) or triacetin. WO 03/053401 describes a formulation which may bothenhance bioavailability and provide controlled releaseof the agentwithin the GI tract. Additional controlled release formulations aredescribed in WO 02/38129, EP 326151, U.S. Pat. No. 5,236,704, WO02/30398, WO 98/13029; U.S. 20030064105, U.S. 20030138488A1, U.S.20030216307A1, U.S. Pat. No. 6,667,060, WO 01/49249, WO 01/49311, WO01/49249, WO 01/49311, and U.S. Pat. No. 5,877,224 materials which mayinclude those described in WO04041195 (including the seal and entericcoating described therein) and pH-sensitive coatings that achievedelivery in the colon including those described in U.S. Pat. No.4,910,021 and WO9001329. U.S. Pat. No. 4,910,021 describes using apH-sensitive material to coat a capsule. WO9001329 describes usingpH-sensitive coatings on beads containing acid, where the acid in thebead core prolongs dissolution of the pH-sensitive coating. U.S. Pat.No. 5,175,003 discloses a dual mechanism polymer mixture composed ofpH-sensitive enteric materials and film-forming plasticizers capable ofconferring permeability to the enteric material, for use indrug-delivery systems; a matrix pellet composed of a dual mechanismpolymer mixture permeated with a drug and sometimes covering apharmaceutically neutral nucleus; a membrane-coated pellet comprising amatrix pellet coated with a dual mechanism polymer mixture envelope ofthe same or different composition; and a pharmaceutical dosage formcontaining matrix pellets. The matrix pellet releases acid-soluble drugsby diffusion in acid pH and by disintegration at pH levels of nominallyabout 5.0 or higher.

The GCC peptides described herein may be formulated in the pH triggeredtargeted control release systems described in WO04052339. The agentsdescribed herein may be formulated according to the methodologydescribed in any of WO03105812 (extruded hyrdratable polymers);WO0243767 (enzyme cleavable membrane translocators); WO03007913 andWO03086297 (mucoadhesive systems); WO02072075 (bilayer laminatedformulation comprising pH lowering agent and absorption enhancer);WO04064769 (amidated polypeptides); WO05063156 (solid lipid suspensionwith pseudotropic and/or thixotropic properties upon melting);WO03035029 and WO03035041 (erodible, gastric retentive dosage forms);U.S. Pat. No. 5,007,790 and U.S. Pat. No. 5,972,389 (sustained releasedosage forms); WO041 1271 1 (oral extended release compositions);WO05027878, WO02072033, and WO02072034 (delayed release compositionswith natural or synthetic gum); WO05030182 (controlled releaseformulations with an ascending rate of release); WO05048998(microencapsulation system); U.S. Pat. No. 5,952,314 (biopolymer); U.S.Pat. No. 5,108,758 (glassy amylose matrix delivery); U.S. Pat. No.5,840,860 (modified starch based delivery). JP10324642 (delivery systemcomprising chitosan and gastric resistant material such as wheat gliadinor zein); U.S. Pat. No. 5,866,619 and U.S. Pat. No. 6,368,629(saccharide containing polymer); U.S. Pat. No. 6,531,152 (describes adrug delivery system containing a water soluble core (Ca pectinate orother water-insoluble polymers) and outer coat which bursts (e.g.hydrophobic polymer-Eudragrit)); U.S. Pat. No. 6,234,464; U.S. Pat. No.6,403,130 (coating with polymer containing casein and high methoxypectin; WO0174 175 (Maillard reaction product); WO05063206 (solubilityincreasing formulation); WO040 19872 (transferring fusion proteins).

The GCC peptides described herein may be formulated usinggastrointestinal retention system technology (GIRES; MerrionPharmaceuticals). GIRES comprises a controlled-release dosage forminside an inflatable pouch, which is placed in a drug capsule for oraladministration. The capsule shell can be a HPMC capsule shell or Gelatincapsule shell. Upon dissolution of the capsule, a gas-generating systeminflates the pouch in the stomach where it is retained for 16-24 hours,all the time releasing agents described herein.

The GCC peptides described herein can also be formulated using the multimatrix system technology (MMX).

The GCC peptides described herein can be formulated in an osmotic deviceincluding the ones disclosed in U.S. Pat. No. 4,503,030, U.S. Pat. No.5,609,590 and U.S. Pat. No. 5,358,502. U.S. Pat. No. 4,503,030 disclosesan osmotic device for dispensing a drug to certain pH regions of thegastrointestinal tract. More particularly, the invention relates to anosmotic device comprising a wall formed of a semi-permeable pH sensitivecomposition that surrounds a compartment containing a drug, with apassageway through the wall connecting the exterior of the device withthe compartment. The device delivers the drug at a controlled rate inthe region of the gastrointestinal tract having a pH of less than 3.5,and the device self-destructs and releases all its drug in the region ofthe gastrointestinal tract having a pH greater than 3.5, therebyproviding total availability for drug absorption. U.S. Pat. Nos.5,609,590 and 5,358,502 disclose an osmotic bursting device fordispensing a beneficial agent to an aqueous environment. The devicecomprises a beneficial agent and osmagent surrounded at least in part bya semi-permeable membrane. The beneficial agent may also function as theosmagent. The semi-permeable membrane is permeable to water andsubstantially impermeable to the beneficial agent and osmagent. Atrigger means is attached to the semi-permeable membrane (e.g., joinstwo capsule halves). The trigger means is activated by a pH of from 3 to9 and triggers the eventual, but sudden, delivery of the beneficialagent. These devices enable the pH-triggered release of the beneficialagent core as a bolus by osmotic bursting.

In one embodiment the formulation contains a GCC agonist peptide,mannitol, silicified microcrystalline cellulose, sodicum starchglycolate, and sodium stearyl fumarate. The GCC agonist is at aconcentration of less than 5% w/w, less than 4%, less than 3% w/w, lessthan 2% w/w, less than 1% w/w, less than 0.5% w/w, or less than 0.25%w/w. In some embodiments the GCC peptide is at a concentration of about0.23% w/w. The GCC peptide is preferably SEQ NO: 1 or SEQ NO: 9. Themannitol is at a concentration of at least 60% w/w, at least 65% w/w, atleast 70% w/w, at least 75% w/w, or at least 80% w/w. In someembodiments the mannitol is present at about 79% w/w (e.g., 79.77%). Themannitol is preferably Mannogem EZ. The silicified microcrystallinecellulose is at a concentration of at least 5% w/w, at least 10% w/w, orat least 15% w/w. In some embodiments the concentration of thesilicified microcrystalline cellulose is about 15% w/w. The silicifiedmicrocrystalline cellulose is preferably Prosolv SMCC 90 LM. The sodicumstarch glycolate is at a concentration of at least 1% w/w, at least 2%w/w, at least 3% w/w, or at least 4% w/w. In some embodiments theconcentration of the sodicum starch glycolate is about 4% w/w. Thesodicum starch glycolate is preferably Explotab. The sodium stearylfumarate is at a concentration of at least 0.2% w/w, at least 0.5% w/w,at least 0.7% w/w, at least 0.8% w/w, at least 0.9, or at least 1% w/w.In some embodiments the concentration of the sodium stearyl fumarate isabout 1% w/w. The sodium stearyl fumarate is preferably Pruv.

In one embodiment the formulation contains a GCC agonist peptide,silicified microcrystalline cellulose, and sodicum starch glycolate. TheGCC agonist is at a concentration of less than 5% w/w, less than 4% w/w,less than 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5%w/w, or less than 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 0.3% w/w. The GCC peptide is preferably SEQ NO: 1or SEQ NO: 9. The silicified microcrystalline cellulose is at aconcentration of at least 10% w/w, at least 20% w/w, at least 30% w/w,at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w,at least 80% w/w, at least 90% w/w, or at least 95% w/w. In someembodiments the concentration of the silicified microcrystallinecellulose is about 95.7% w/w. The silicified microcrystalline celluloseis preferably Prosolv SMCC 90 HD. The sodicum starch glycolate is at aconcentration of at least 1% w/w, at least 2% w/w, at least 3% w/w, orat least 4% w/w. In some embodiments the concentration of the sodicumstarch glycolate is 4% w/w. The sodicum starch glycolate is preferablyExplotab.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, calcium chloride dihydrate, leucine, andhyrpomellose. The GCC agonist is at a concentration of less than 5% w/w,less than 4% w/w, less than 3% w/w, less than 2% w/w, less than 1% w/w,less than 0.5% w/w, or less than 0.25% w/w. In some embodiments the GCCpeptide is at a concentration of about 0.3246% w/w. The GCC peptide ispreferably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cellulose is ata concentration of at least 50% w/w, at least 60% w/w, at least 70% w/w,at least 80% w/w, at least 90% w/w, at least 95% w/w, or at least 99%w/w. In some embodiments the concentration of the microcrystallinecellulose is about 99.10% w/w. The microcrystalline cellulose ispreferably Celphere SCP-100. The calcium chloride dihydrate is at aconcentration of at least 0.1% w/w, at least 0.15% w/w, at least 0.2%w/w, or at least 0.25% w/w. In some embodiments the concentration of thecalcium chloride dihydrate is about 0.2622% w/w. The leucine is at aconcentration of at least 0.05% w/w, at least 0.1% w/w, at least 0.12%w/w, or at least 0.15% w/w. In some embodiments the concentration ofleucine is about 0.12% w/w. The hypromellose is at a concentration of atleast 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or at least 0.25%w/w. In some embodiments the concentration of the hypromellose is about0.2% w/w. The hypromellose is preferably Methocel E5 PremLV.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, calcium chloride dihydrate, leucine,hypromellose, and magnesium stearate. The GCC agonist is at aconcentration of less than 5% w/w, less than 4% w/w, less than 3% w/w,less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or less than0.25% w/w. In some embodiments the GCC peptide is at a concentration ofabout 0.36% w/w. The GCC peptide is preferably SEQ NO: 1 or SEQ NO: 9.The microcrystalline cellulose is at a concentration of at least 50%w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90%w/w, at least 95% w/w, or at least 99% w/w. In some embodiments theconcentration of the microcrystalline cellulose is about 98.75% w/w. Themicrocrystalline cellulose is preferably Avicel PH 102. The calciumchloride dihydrate is at a concentration of at least 0.1% w/w, at least0.15% w/w, at least 0.2% w/w, at least 0.25% w/w, or at least 0.3% w/w.In some embodiments the concentration of the calcium chloride dihydrateis about 0.29% w/w. The leucine is at a concentration of at least 0.05%w/w, at least 0.1% w/w, at least 0.12% w/w, or at least 0.15% w/w. Insome embodiments the concentration of leucine is about 0.13% w/w. Thehypromellose is at a concentration of at least 0.1% w/w, at least 0.15%w/w, at least 0.2% w/w, or at least 0.25% w/w. In some embodiments theconcentration of the hypromellose is about 0.22% w/w. The hypromelloseis preferably Methocel E5 PremLV. The magnesium stearate is at aconcentration of at least 0.1% w/w, at least 0.15% w/w, at least 0.2%w/w, or at least 0.25% w/w. In some embodiments the concentration of themagnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, and magnesium stearate. The GCC agonist isat a concentration of less than 5% w/w, less than 4% w/w, less than 3%w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or lessthan 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 0.32% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The microcrystalline cellulose is at a concentration ofat least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w,at least 90% w/w, at least 95% w/w, or at least 99% w/w. In someembodiments the concentration of the microcrystalline cellulose is about99.43% w/w. The microcrystalline cellulose is preferably Avicel PH 102.The magnesium stearate is at a concentration of at least 0.1% w/w, atleast 0.15% w/w, at least 0.2% w/w, or at least 0.25% w/w. In someembodiments the concentration of the magnesium stearate is about 0.25%w/w.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, and magnesium stearate. The GCC agonist isat a concentration of less than 5% w/w, less than 4% w/w, less than 3%w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or lessthan 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 0.32% w/w, about 1.18% w/w. The GCC peptide ispreferably SEQ NO: 1 or SEQ NO: 9. The microcrystalline cellulose is ata concentration of at least 30% w/w, at least 40% w/w, at least 50% w/w,at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w,at least 95% w/w, or at least 99% w/w. In some embodiments theconcentration of the microcrystalline cellulose is about 98.57% w/w. Themicrocrystalline cellulose is preferably Avicel PH 102. The magnesiumstearate is at a concentration of at least 0.1% w/w, at least 0.15% w/w,at least 0.2% w/w, or at least 0.25% w/w. In some embodiments theconcentration of the magnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, and magnesium stearate. The GCC agonist isat a concentration of less than 5% w/w, less than 4% w/w, less than 3%w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or lessthan 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 1.18% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The microcrystalline cellulose is at a concentration ofat least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w,at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w,or at least 99% w/w. In some embodiments the concentration of themicrocrystalline cellulose is about 97.09% w/w. The microcrystallinecellulose is preferably Avicel PH 112. The magnesium stearate is at aconcentration of at least 0.1% w/w, at least 0.15% w/w, at least 0.2%w/w, or at least 0.25% w/w. In some embodiments the concentration of themagnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,trehalose granules, hypromellose, histidine, calcium ascorbate,trehalose powder, microcrystalline cellulose, and magnesium stearate.The GCC agonist is at a concentration of less than 5% w/w, less than 4%w/w, less than 3% w/w, less than 2% w/w, less than 1% w/w, less than0.5% w/w, or less than 0.25% w/w. In some embodiments the GCC peptide isat a concentration of about 1.18% w/w. The GCC peptide is preferably SEQNO: 1 or SEQ NO: 9. The trehalose granules are at a concentration of atleast 50% w/w, at least 55% w/w, at least 60% w/w, at least 65% w/w, atleast 70% w/w, or at least 75% w/w. In some embodiments theconcentration of the trehalose granules is 55-75% w/w. In a particularembodiment, the concentration of the trehalose granules is 70.48% w/w.The hypromellose is at a concentration of at least 0.1% w/w, at least0.2% w/w, at least 0.3% w/w, at least 0.4% w/w, or at least 0.5% w/w. Insome embodiments the concentration of the hypromellose is 0.2-2% w/w. Ina particular embodiment the concentration of the hypromellose about 0.5%w/w. The hypromellose is preferably Methocel ES Premium LV. The histineis a concentration of at least 0.6% w/w, at least 0.8% w/w, at least0.9% w/w, at least 1% w/w, at least 3% w/w, or at least 5% w/w. In someembodiments the concentration of the histidine is 1-6% w/w. In aparticular embodiment, the concentration of the arginine is 1.48% w/w.The calcium ascorbate is at a concentration of at least 0.05% w/w, atleast 0.07% w/w, at least 0.09% w/w, or at least 0.1% w/w. In someembodiments the concentration of the calcium ascorbate is 0.05-10% w/w.In a particular embodiment, the concentration of the calcium ascorbateis about 0.1% w/w. The trehalose powder is at a concentration of atleast 0.5% w/w, at least 0.7% w/w, at least 0.8% w/w, at least 0.9% w/w,at least 1% w/w, or at least 1.2% w/w. In some embodiments theconcentration of the trehalose powder is 0.5-4% w/w. In a particularembodiment, the concentration of the trehalose powder is 1.02% w/w. Themicrocrystalline cellulose is at a concentration of at least 10% w/w, atleast 20% w/w, or at least 25% w/w. In some embodiments theconcentration of the microcrystalline cellulose is 20-40% w/w. In aparticular embodiment, the concentration of the microcrystallinecellulose is 25% w/w. The microcrystalline cellulose is preferablyAvicel PH 200. The magnesium stearate is at a concentration of at least0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or at least 0.25% w/w.In some embodiments the concentration of the magnesium stearate is0.2-1% w/w. In a particular embodiment the concentration of themagnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,trehalose granules, hypromellose, arginine, calcium ascorbate, trehalosepowder, microcrystalline cellulose, and magnesium stearate. The GCCagonist is at a concentration of less than 5% w/w, less than 4% w/w,less than 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5%w/w, or less than 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 1.17% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The trehalose granules are at a concentration of atleast 50% w/w, at least 55% w/w, at least 60% w/w, at least 65% w/w, atleast 70% w/w, or at least 75% w/w. In some embodiments theconcentration of the trehalose granules is 55-75% w/w. In a particularembodiment, the concentration of the trehalose granules is 70.31% w/w.The hypromellose is at a concentration of at least 0.1% w/w, at least0.2% w/w, at least 0.3% w/w, at least 0.4% w/w, or at least 0.5% w/w. Insome embodiments the concentration of the hypromellose is 0.2-2% w/w. Ina particular embodiment the concentration of the hypromellose about 0.5%w/w. The hypromellose is preferably Methocel ES Premium LV. The arginineis a concentration of at least 0.5% w/w, at least 1% w/w, at least 1.5%w/w, or at least 2% w/w. In some embodiments the concentration of thearginine is 1-6% w/w. In a particular embodiment, the concentration ofthe arginine is 1.66% w/w. The calcium ascorbate is at a concentrationof at least 0.05% w/w, at least 0.07% w/w, at least 0.09% w/w, or atleast 0.1% w/w. In some embodiments the concentration of the calciumascorbate is 0.05-10% w/w. In a particular embodiment, the concentrationof the calcium ascorbate is about 0.1% w/w. The trehalose powder is at aconcentration of at least 0.5% w/w, at least 0.7% w/w, at least 0.8%w/w, at least 0.9% w/w, at least 1% w/w, or at least 1.2% w/w. In someembodiments the concentration of the trehalose powder is 0.5-4% w/w. Ina particular embodiment, the concentration of the trehalose powder is1.02% w/w. The microcrystalline cellulose is at a concentration of atleast 10% w/w, at least 20% w/w, or at least 25% w/w. In someembodiments the concentration of the microcrystalline cellulose is20-40% w/w. In a particular embodiment, the concentration of themicrocrystalline cellulose is 25% w/w. The microcrystalline cellulose ispreferably Avicel PH 200. The magnesium stearate is at a concentrationof at least 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or at least0.25% w/w. In some embodiments the concentration of the magnesiumstearate is 0.2-1% w/w. In a particular embodiment the concentration ofthe magnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,trehalose granules, hypromellose, TRIS, calcium ascorbate, trehalosepowder, microcrystalline cellulose, and magnesium stearate. The GCCagonist is at a concentration of less than 5% w/w, less than 4% w/w,less than 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5%w/w, or less than 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 1.17% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The trehalose granules are at a concentration of atleast 50% w/w, at least 55% w/w, at least 60% w/w, at least 65% w/w, atleast 70% w/w, or at least 75% w/w. In some embodiments theconcentration of the trehalose granules is 55-75% w/w. In a particularembodiment, the concentration of the trehalose granules is 70.81% w/w.The hypromellose is at a concentration of at least 0.1% w/w, at least0.2% w/w, at least 0.3% w/w, at least 0.4% w/w, or at least 0.5% w/w. Insome embodiments the concentration of the hypromellose is 0.2-2% w/w. Ina particular embodiment the concentration of the hypromellose about 0.5%w/w. The hypromellose is preferably Methocel ES Premium LV. The TRIS isa concentration of at least 0.6% w/w, at least 0.8% w/w, at least 0.9%w/w, or at least 1% w/w. In some embodiments the concentration of theTRIS is 0.5-6% w/w. In a particular embodiment, the concentration of thearginine is 1.15% w/w. The calcium ascorbate is at a concentration of atleast 0.05% w/w, at least 0.07% w/w, at least 0.1% w/w, or at least 1%w/w. In some embodiments the concentration of the calcium ascorbate is0.05-10% w/w. In a particular embodiment, the concentration of thecalcium ascorbate is about 0.1% w/w. The trehalose powder is at aconcentration of at least 0.5% w/w, at least 0.7% w/w, at least 0.8%w/w, at least 0.9% w/w, at least 1% w/w, or at least 1.2% w/w. In someembodiments the concentration of the trehalose powder is 0.5-4% w/w. Ina particular embodiment, the concentration of the trehalose powder is1.02% w/w. The microcrystalline cellulose is at a concentration of atleast 10% w/w, at least 20% w/w, or at least 25% w/w. In someembodiments the concentration of the microcrystalline cellulose is20-40% w/w. In a particular embodiment, the concentration of themicrocrystalline cellulose is 25% w/w. The microcrystalline cellulose ispreferably Avicel PH 200. The magnesium stearate is at a concentrationof at least 0.1% w/w, at least 0.15% w/w, at least 0.2% w/w, or at least0.25% w/w. In some embodiments the concentration of the magnesiumstearate is 0.2-1% w/w. In a particular embodiment the concentration ofthe magnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, and magnesium stearate. The GCC agonist isat a concentration of less than 5% w/w, less than 4% w/w, less than 3%w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or lessthan 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 1.10% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The microcrystalline cellulose is at a concentration ofat least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w,at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w,or at least 99% w/w. In some embodiments the concentration of themicrocrystalline cellulose is about 98.64% w/w. The microcrystallinecellulose is preferably Avicel PH 102. The magnesium stearate is at aconcentration of at least 0.1% w/w, at least 0.15% w/w, at least 0.2%w/w, or at least 0.25% w/w. In some embodiments the concentration of themagnesium stearate is about 0.25% w/w.

In one embodiment the formulation contains a GCC agonist peptide,microcrystalline cellulose, and magnesium stearate. The GCC agonist isat a concentration of less than 5% w/w, less than 4% w/w, less than 3%w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, or lessthan 0.25% w/w. In some embodiments the GCC peptide is at aconcentration of about 3.32% w/w. The GCC peptide is preferably SEQ NO:1 or SEQ NO: 9. The microcrystalline cellulose is at a concentration ofat least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w,at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w,or at least 99% w/w. In some embodiments the concentration of themicrocrystalline cellulose is about 96.43% w/w. The microcrystallinecellulose is preferably Avicel PH 102. The magnesium stearate is at aconcentration of at least 0.1% w/w, at least 0.15% w/w, at least 0.2%w/w, or at least 0.25% w/w. In some embodiments the concentration of themagnesium stearate is about 0.25% w/w.

1.2 GCC Agonists

The GCC agonists for use in the formulations and methods of theinvention bind to guanylate cyclase C and stimulate intracellularproduction of cGMP. Optionally, the GCC agonists induce apoptosis andinhibit proliferation of epithelial cells. The term, “guanylate cyclaseC” refers to a transmembrane form of guanylate cyclase that acts as theintestinal receptor for the heat-stable toxin (ST) peptides secreted byenteric bacteria. Guanylate cyclase C is also the receptor for thenaturally occurring peptides guanylin and uroguanylin. The possibilitythat there may be different receptors for each of these peptides has notbeen excluded. Hence, the term “guanylate cyclase C” may also encompassa class of transmembrane guanylate cyclase receptors expressed onepithelial cells lining the gastrointestinal mucosa.

The term “GCC agonist” refers to both peptides and non-peptide compoundssuch as that bind to an intestinal guanylate cyclase C and stimulate theintracellular production of cGMP. Where the GCC agonist is a peptide,the term encompasses biologically active fragments of such peptides andpro-peptides that bind to guanylate cyclase C and stimulate theintracellular production of cGMP.

Preferably, the GCC agonists for use in the formulations and methods ofthe invention stimulate intracellular cGMP production at higher levelsthan naturally occurring GCC agonists such as uroguanylin, guanylin, andST peptides. In some embodiments, the GCC agonists stimulateintracellular cGMP production at higher levels than the peptidedesignated SP-304 (SEQ ID NO:1). In specific embodiments, a GCC agonistfor use in the formulations and methods of the invention stimulates 5%,10%, 20%, 30%, 40%, 50%, 75%, 90% or more intracellular cGMP compared touroguanylin, guanylin, lymphoguanylin, linaclotide, ST peptides, orSP-304. The terms “induce” and “stimulate” are used interchangeablythroughout the specification.

Preferably, the GCC agonists for use in the formulations and methods ofthe invention are more stable than naturally occurring GCC agonists suchas uroguanylin, guanylin, and ST peptides. In some embodiments, the GCCagonists are more stable than the peptide designated SP-304. “Stability”in this context refers to resistance to degradation in gastrointestinalfluid and/or intestinal fluid (or simulated gastrointestinal orintestinal fluids) compared to the reference peptide. For example, theGCC agonists for use in the formulations and methods of the inventionpreferably degrade 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% orless compared to naturally occurring GCC angonists and/or SP-304.

The GCC agonists for use in the formulations and methods of theinvention are preferably peptides. In some embodiments, the GCC agonistpeptide is less than 30 amino acids in length. In particularembodiments, the GCC agonist peptide is less than or equal to 30, 25,20, 15, 14, 13, 12, 11, 10, or 5 amino acids in length. Examples of GCCagonist peptides for use in the formulations and methods of theinvention include those described in U.S. Ser. No. 12/133,344, filedJun. 4, 2008, Ser. No. 12/478,505, filed Jun. 4, 2009; Ser. No.12/478,511, filed Jun. 4, 2009; Ser. No. 12/504,288, filed Jul. 16,2009; and U.S. Provisional Application Ser. No. 60/933,194, filed Jun.4, 2007; 61/058,888, filed Jun. 4, 2008; 61/058,892, filed Jun. 4, 2008;and 61/081,289, filed Jul. 16, 2008, each of which is incorporated byreference herein in its entirety.

Specific examples of GCC agonist peptides for use in the formulationsand methods of the invention include those described in Tables I-VIIbelow. As used Tables I-VII, the terms “PEG3” or “3PEG” refer to apolyethylene glycol such as aminoethyloxy-ethyloxy-acetic acid (AeeA),and polymers thereof. The term “X_(aa)” refers to any natural orunnatural amino acid or amino acid analogue. The term “M_(aa)” refers toa cysteine (Cys), penicillamine (Pen) homocysteine, or3-mercaptoproline. The term “Xaa_(n1)” is meant to denote an amino acidsequence of any natural or unnatural amino acid or amino acid analoguethat is one, two or three residues in length; Xaa_(n2) is meant todenote an amino acid sequence that is zero or one residue in length; andXaa_(n3) is meant to denote an amino acid sequence zero, one, two,three, four, five or six residues in length. Additionally, any aminoacid represented by Xaa, Xaa_(n1), Xaa_(n2), or Xaa_(n3) may be anL-amino acid, a D-amino acid, a methylated amino acid or any combinationof thereof. Optionally, any GCC agonist peptide represented by FormulasI to XX in the tables may contain on or more polyethylene glycolresidues at the N-terminus, C-terminus or both.

In certain embodiments, a GCC agonist formulation of the inventioncomprises a peptide selected from SEQ ID NOs: 1-249, the sequences ofwhich are set forth below in Tables I to VII below. In one embodiment, aGCC agonist formulation comprises the peptide designated by SEQ IDNOs:1, 8, 9, 55, or 56.

In certain embodiments, a GCC agonist formulation of the inventioncomprises a peptide that is substantially equivalent to a peptideselected from SEQ ID NOs: 1-249. The term “substantially equivalent”refers to a peptide that has an amino acid sequence equivalent to thatof the binding domain where certain residues may be deleted or replacedwith other amino acids without impairing the peptide's ability to bindto an intestinal guanylate cyclase receptor and stimulate fluid andelectrolyte transport.

1.2.1 GCC Agonist Peptides

In a preferred embodiment, the GCC agonists for use in the formulationsand methods of the invention are GCC agonist peptides. In certainembodiments, the GCC agonist peptides are analogues of uroguanylin or abacterial ST peptide. Uroguanylin is a circulating peptide hormone withnatriuretic activity. An ST peptide is a member of a family of heatstable enterotoxins (ST peptides) secreted by pathogenic strains of E.coli and other enteric bacteria that activate guanylate cyclase receptorand cause secretory diarrhea. Unlike bacterial ST peptides, the bindingof uroguanylin to guanylate cyclase receptor is dependent on thephysiological pH of the gut. Therefore, uroguanylin is expected toregulate fluid and electrolyte transport in a pH dependent manner andwithout causing severe diarrhea.

The GCC agonist peptides for use in the formulations and methods of theinvention can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into a D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence.

The GCC agonist peptides for use in the formulations and methods of theinvention are able to induce intracellular cGMP production in cells andtissues expressing guanylate cyclase C. In certain embodiments, the GCCagonist peptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to naturally occurring GCC agonists such asuroguanylin, guanylin, or ST peptides. Optionally, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared SP-304 (SEQ ID NO:1). In furtherembodiments, the GCC agonist peptide stimulates apoptosis, e.g.,programmed cell death, or activate the cystic fibrosis transmembraneconductance regulator (CFTR).

In some embodiments, the GCC agonist peptides for use in theformulations and methods of the invention are more stable than naturallyoccurring GCC agonists and/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide)(SEQ ID NO: 55) or SP-340 (SEQ ID NO: 56). For example, the GCC agonistpeptide degrades 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90% orless compared to naturally occurring GCC agonists and/or SP-304, SP-339(linaclotide) or SP-340. In certain embodiments, the GCC agonistpeptides for use in the formulations and methods of the invention aremore stable to proteolytic digestion than naturally occurring GCCagonists and/or SP-304 (SEQ ID NO:1), SP-339 (linaclotide) (SEQ ID NO:55) or SP-340 (SEQ ID NO: 56). In one embodiment, a GCC agonist peptideis pegylated in order to render the peptides more resistant towardsprotealysis by enzymes of the gastrointestinal tract. In a preferredembodiment, the GCC agonist peptide is pegylated with theaminoethyloxy-ethyloxy-acetic acid (Aeea) group at its C-terminal end,at its N-terminal end, or at both termini.

Specific examples of GCC agonist peptides that can be used in themethods and formulations of the invention include a peptide selectedfrom the group designated by SEQ ID NOs: 1-249.

In one embodiment, the GCC agonist peptide is a peptide having the aminoacid sequence of any one of Formulas X-XVII (e.g. SEQ ID NO:87-98).

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula I, wherein at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at positions 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn¹, Asp² orGlu³ (or a combination thereof) of Formula I is a D-amino acid or amethylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula II, wherein at least one amino acidof Formula II is a D-amino acid or a methylated amino acid. Preferably,the amino acid denoted by Xaa_(n2) of Formula II is a D-amino acid or amethylated amino acid. In some embodiments, the amino acid denoted byXaa_(n2) of Formula II is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more amino acids denoted by Xaa_(n1) ofFormula II is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position Xaa⁶ of Formula II is a leucine, a serine, or atyrosine.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula III, wherein at least one amino acid ofFormula III is a D-amino acid or a methylated amino acid and/or Maa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III is a D-amino acid ora methylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine.

In other embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula IV, wherein at least one amino acid ofFormula IV is a D-amino acid or a methylated amino acid, and/or Maa isnot a cysteine. Preferably, the Xaa_(n2) of Formula IV is a D-amino acidor a methylated amino acid. In some embodiments, the amino acid denotedby Xaa_(n2) of Formula IV is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula IV is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, aserine, or a tyrosine.

In further embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula V, wherein at at least one amino acid ofFormula V is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid. For example, the amino acid at position 16 (i.e., Xaa¹⁶) ofFormula V is a d-leucine or a d-serine. Optionally, one or more of theamino acids at position 1-3 of Formula V are D-amino acids or methylatedamino acids or a combination of D-amino acids or methylated amino acids.For example, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula Vis a D-amino acids or a methylated amino acid. Preferably, the aminoacid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula VI, VII, VIII, or IX. Preferably, theamino acid at position 6 of Formula VI, VII, VIII, or IX is a leucine, aserine, or a tyrosine. In some aspects the amino acid at position 16 ofFormula VI, VII, VIII, or IX is a leucine or a serine. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI orXVII. Optionally, one or more amino acids of Formulas X, XI, XII, XIII,XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid.Preferably, the amino acid at the carboxy terminus of the peptidesaccording to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is aD-amino acid or a methylated amino acid. For example the amino acid atthe carboxy terminus of the peptides according to Formulas X, XI, XII,XIII, XIV, XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenyalanine or a serine. Most preferably the amino acid denoted by Xaa⁶of Formula XIV is a phenyalanine or a serine. Preferably, the amino aciddenoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenyalanine, or a serine. Most preferably, the amino acid position Xaa⁴of Formula V, XVI or XVII is a phenyalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 a glutamic acid. Preferably, the amino acid at position 6 ofFormula XVIII is an isoleucine, valine, serine, threonine or tyrosine.Preferably, the amino acid at position 8 of Formula XVIII is a valine orisoleucine. Preferably, the amino acid at position 9 of Formula XVIII isa an asparagine. Preferably, the amino acid at position 10 of FormulaXVIII is a valine or an methionine. Preferably, the amino acid atposition 11 of Formula XVIII is an alanine. Preferably, the amino acidat position 13 of Formula XVIII is a threonine. Preferably, the aminoacid at position 14 of Formula XVIII is a glycine. Preferably, the aminoacid at position 16 of Formula XVIII is a leucine, serine or threonine.

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine or tyrosine.Preferably, the amino acid at position 8, 10, 11, or 13 of Formula XIXis a alanine. Preferably, the amino acid at position 9 of Formula XIX isan asparagine or a phenylalanine. Preferably, the amino acid at position14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or a aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid. Preferably, the amino acid at position 6 of Formula XX isthreonine, glutamine, tyrosine, isoleucine, or leucine. Preferably, theamino acid at position 8 of Formula XX is isoleucine or valine.Preferably, the amino acid at position 9 of Formula XX is asparagine.Preferably, the amino acid at position 10 of Formula XX is methionine orvaline. Preferably, the amino acid at position 11 of Formula XX isalanine. Preferably, the amino acid at position 13 of Formula XX is athreonione. Preferably, the amino acid at position 1 of Formula XX is aglycine. Preferably, the amino acid at position 15 of Formula XX is atyrosine. Optionally, the amino acid at position 15 of Formula XX is twoamino acid in length and is Cysteine (Cys), Penicillamine (Pen)homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the GCC agonistpeptides are replaced by a non-naturally occurring amino acid or anaturally or non-naturally occurring amino acid analog. Such amino acidsand amino acid analogs are known in the art. See, for example, Hunt,“The Non-Protein Amino Acids,” in Chemistry and Biochemistry of theAmino Acids, Barrett, Chapman and Hall, 1985. In some embodiments, anamino acid is replaced by a naturally-occurring, non-essential aminoacid, e.g., taurine. Non-limiting examples of naturally occurring aminoacids that can be replaced by non-protein amino acids include thefollowing: (1) an aromatic amino acid can be replaced by3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg andnorTyr and other amino acids including Phe and Tyr can be substitutedby, e.g., a halogen, —CH3, —OH, —CH2NH3, —C(O)H, —CH2CH3, —CN,—CH2CH2CH3, —SH, or another group; (3) glutamine residues can besubstituted with gamma-Hydroxy-Glu or gamma-Carboxy-Glu; (4) tyrosineresidues can be substituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp; (5) prolineresidues can be substituted with homopro (L-pipecolic acid);hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3; and (6) alanine residues can be substituted withalpha-substitued or N-methylated amino acid such as alpha-aminoisobutyric acid (aib), L/D-alpha-ethylalanine (L/D-isovaline),L/D-methylvaline, or L/D-alpha-methylleucine or a non-natural amino acidsuch as beta-fluoro-Ala. Alanine can also be substituted with: n=0, 1,2, 3 Glycine residues can be substituted with alpha-amino isobutyricacid (aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of non-natural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc β-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, 0-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,US20060019347 (paragraphs 410-418) and the references cited therein. Thepolypeptides of the invention can include further modificationsincluding those described in US20060019347, paragraph 589. Exempary GCCagonist peptides which include a non-naturally occurring amino acidinclude for example SP-368 and SP-369.

In some embodiments, the GCC agonist peptides are cyclic peptides. GCCagonist cyclic peptides can be prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K3Fe(CN)₆ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In various embodiments, the GCC agonist peptides are[4,12; 7,15] bicycles.

In certain embodiments, one or both Cys residues which normally form adisulfide bond in a GCC agonist peptide are replaced with homocysteine,penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int. J. Pept.Protein Res. 48:274), β, β dimethylcysteine (Hunt et al. 1993 Int. J.Pept. Protein Res. 42:249), or diaminopropionic acid (Smith et al. 1978J. Med. Chem. 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In certain embodiments, one or more disulfide bonds in a GCC agonistpeptide are replaced by alternative covalent cross-links, e.g., an amidelinkage (—CH₂CH(O)NHCH₂— or —CH₂NHCH(O)CH₂—), an ester linkage, athioester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage,a thiourea linkage, a phosphonate ester linkage, an alkyl linkage(—CH₂CH₂CH₂CH₂—), an alkenyl linkage (—CH₂CH═CHCH₂—), an ether linkage(—CH₂CH₂OCH₂— or —CH₂OCH₂CH₂—), a thioether linkage (—CH₂CH₂SCH₂— or—CH₂SCH₂CH₂—), an amine linkage (—CH₂CH₂NHCH₂— or —CH₂NHCH₂CH₂—) or athioamide linkage (—CH₂CH(S)HNHCH₂— or —CH₂NHCH(S)CH₂—). For example,Ledu et al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describe methodsfor preparing lactam and amide cross-links. Exemplary GCC agonistpeptides which include a lactam bridge include, for example, SP-370.

In certain embodiments, the GCC agonist peptides have one or moreconventional polypeptide bonds replaced by an alternative bond. Suchreplacements can increase the stability of the polypeptide. For example,replacement of the polypeptide bond between a residue amino terminal toan aromatic residue (e.g. Tyr, Phe, Trp) with an alternative bond canreduce cleavage by carboxy peptidases and may increase half-life in thedigestive tract. Bonds that can replace polypeptide bonds include: aretro-inverso bond (C(O)—NH instead of NH—C(O); a reduced amide bond(NH—CH₂); a thiomethylene bond (S—CH₂ or CH₂—S); an oxomethylene bond(O—CH₂ or CH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond(C(S)—NH); a trans-olefine bond (CH═CH); a fiuoro substitutedtrans-olefme bond (CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O)wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH₃.

In certain embodiments, the GCC agonist peptides are modified usingstandard modifications. Modifications may occur at the amino (N—),carboxy (C—) terminus, internally or a combination of any of thepreceeding. In one aspect described herein, there may be more than onetype of modification on the polypeptide. Modifications include but arenot limited to: acetylation, amidation, biotinylation, cinnamoylation,farnesylation, formylation, myristoylation, palmitoylation,phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,sulfurylation and cyclisation (via disulfide bridges or amidecyclisation), and modification by Cys3 or Cys5. The GCC agonist peptidesdescribed herein may also be modified by 2, 4-dinitrophenyl (DNP),DNP-lysine, modification by 7-Amino-4-methyl-coumarin (AMC),flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide,rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid),dabcyl, dabsyl, dansyl, texas red, FMOC, and Tamra(Tetramethylrhodamine). The GCC agonist peptides described herein mayalso be conjugated to, for example, polyethylene glycol (PEG); alkylgroups (e.g., C1-C20 straight or branched alkyl groups); fatty acidradicals; combinations of PEG, alkyl groups and fatty acid radicals(See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations inPharmaceutical Technology 106-110); BSA and KLH (Keyhole LimpetHemocyanin). The addition of PEG and other polymers which can be used tomodify polypeptides of the invention is described in US20060 19347section IX.

A GCC agonist peptide can also be a derivatives of a GCC agonist peptidedescribed herein. For example, a derivative includes hybrid and modifiedforms of GCC agonist peptides in which certain amino acids have beendeleted or replaced. A modification may also include glycosylation.Preferrably, where the modification is an amino acid substitution, it isa conservative substitution at one or more positions that are predictedto be non-essential amino acid residues for the biological activity ofthe peptide. A “conservative substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

In one embodiment, a GCC agonist peptide described herein is subjectedto random mutagenesis in order to identify mutants having biologicalactivity.

In one embodiment, the GCC agonist peptide is substantially homologousis a GCC agonist peptide described herein. Such substantially homologouspeptides can be isolated by virtue of cross-reactivity with antibodiesto a GCC agonist peptide described herein.

Further examples of GCC agonist peptides that can be used in the methodsand formulations of the invention are found in Tables I-VII below.

1.2.2 Preparation of GCC Agonist Peptides

GCC agonist peptides can be prepared using art recognized techniquessuch as molecular cloning, peptide synthesis, or site-directedmutagenesis.

Peptide synthesis can be performed using standard solution phase orsolid phase peptide synthesis techniques or a combination of bothprocess where segments are synthesized by solid phase and condensed insolution phase, in which a peptide linkage occurs through the directcondensation of the amino group of one amino acid with the carboxy groupof the other amino acid with the elimination of a water molecule.Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents must permit their ready removal, without inducing breakdownof the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor. Solidphase peptide synthesis uses an insoluble polymer for support duringorganic synthesis. The polymer-supported peptide chain permits the useof simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc., 1963, 85:2149, which involves assembling a linearpeptide chain on a resin support using protected amino acids. Solidphase peptide synthesis typically utilizes either the Boc or Fmocstrategy, which are well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the synthesis.In addition, solid phase synthesis is generally most suitable whenpeptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Alternatively the GCC agonist peptides are produced by modern cloningtechniques For example, the GCC agonist peptides are produced either inbacteria including, without limitation, E. coli, or in other existingsystems for polypeptide or protein production (e.g., Bacillus subtilis,baculovirus expression systems using Drosophila Sf9 cells, yeast orfilamentous fungal expression systems, mammalian cell expressionsystems), or they can be chemically synthesized. If the GCC agonistpeptide or variant peptide is to be produced in bacteria, e.g., E. coli,the nucleic acid molecule encoding the polypeptide may also encode aleader sequence that permits the secretion of the mature polypeptidefrom the cell. Thus, the sequence encoding the polypeptide can includethe pre sequence and the pro sequence of, for example, anaturally-occurring bacterial ST polypeptide. The secreted, maturepolypeptide can be purified from the culture medium.

The sequence encoding a GCC agonist peptide described herein can beinserted into a vector capable of delivering and maintaining the nucleicacid molecule in a bacterial cell. The DNA molecule may be inserted intoan autonomously replicating vector (suitable vectors include, forexample, pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleicacid may be a bacterial or bacteriophage DNA such as bacteriophagelambda or M13 and derivatives thereof. Construction of a vectorcontaining a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include but are not limited to, E. coli, B subtilis, Pseudomonas,Salmonella. The genetic construct also includes, in addition to theencoding nucleic acid molecule, elements that allow expression, such asa promoter and regulatory sequences. The expression vectors may containtranscriptional control sequences that control transcriptionalinitiation, such as promoter, enhancer, operator, and repressorsequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCC agonist peptidedescribed herein can also be fused to a nucleic acid encoding apolypeptide affinity tag, e.g., glutathione S-transferase (GST), maltoseE binding protein, protein A, FLAG tag, hexa-histidine, myc tag or theinfluenza HA tag, in order to facilitate purification. The affinity tagor reporter fusion joins the reading frame of the polypeptide ofinterest to the reading frame of the gene encoding the affinity tag suchthat a translational fusion is generated. Expression of the fusion generesults in translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCC agonist peptides and variants described hereinin protein expression systems other than bacteria, and well known tothose skilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

TABLE I GCRA Peptides (SP-304 and Derivatives) Position of SEQ ID NameDisulfide bonds Structure NO SP-304 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶1 SP-326 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵2 SP-327 C2:C10, C5:C13Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴3 SP-328 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴4 SP-329 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 5SP-330 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³ 6SP-331 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹² 7 SP332C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶8 SP-333 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶9 SP-334 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶10 SP-335 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶11 SP-336 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶12 SP-337 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶13 SP-338 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵14 SP-342 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG315 SP-343 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG316 SP-344 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG317 SP-347 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG318 SP-348 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶19 SP-350 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶20 SP-352 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG321 SP-358 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶⁻22 PEG3 SP-359 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶23 SP-360 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶⁻PEG324 SP-361 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG325 SP-362 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶26 SP-368 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶27 SP-369 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶28 SP-370 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹29 SP-371 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶30 SP-372 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶31 N1 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG332 N2 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶33 N3 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶PEG3 34 N4 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG335 N5 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶36 N6 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG337 N7 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶38 N8 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG339 N9 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶40 N10 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG341 N11 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG342 N12 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶43 N13 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG344 Formula I C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶45 Formula II C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2)¹⁶ 46 Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaa_(n2)47 III Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2)48 IV Formula V C4:C12, C7:C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶49 Formula C4:C12, C7:C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-X3⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶50 VI Formula C4:C12, C7:C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶51 VII Formula C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶52 VII Formula C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶53 VIII Formula C4:C12, C7:C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶54 IX

TABLE II Linaclotide and Derivatives Position of Disulfide SEQ ID Namebonds Structure NO: SP-339 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 (linaclotide) SP-340 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1:C6, C2:C10, C5:13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG357 SP-353 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶58 SP-354 C3:C8, C4:C12, C7:15Asn¹-Phe²⁻Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶59 SP-355 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1:C6, C2:C10, C5:13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶62 SP-375 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶63 SP-376 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶64 SP-377 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶65 SP-378 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶66 SP-379 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶67 SP-380 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶68 SP-381 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶69 SP-382 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶70 SP-383 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶71 SP384 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1:C6, C2:C10, C5:13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1:C6, C2:C10, C5:13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-76 Tyr¹⁶-PEG3 N18 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-77 Tyr¹⁶ N19 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-78 PEG3 N20 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-79 Tyr¹⁶-PEG3 N21 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-80 Tyr¹⁶ N22 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-81 PEG3 N23 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-82 Tyr¹⁶-PEG3 N24 C3:C8, C4:C12, C7:15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-83 Tyr¹⁶ N25 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-84 PEG3 N26 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1:C6, C2:C10, C5:13Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1:6, 2:10, 5:13Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 N31 1:6, 2:10, 5:13Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9:C14, C10:C18,Xaa¹-Xaa²⁻Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-91 C13:21 Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XI C9:C14,C10:C18,Xaa¹-Xaa²⁻Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-92 C13:21 Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XII C3:C8, C4:C12,C7:15Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹⁻Cys¹²-Xaa³-Xaa¹⁴⁻Cys¹⁵-Xaa¹⁶93 Formula 3:8, 4:12, C:15Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷⁻Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹⁻Cys12-Xaa¹³-Xaa¹⁴⁻Cys¹⁵-94 XIII Xaa¹⁶ Formula 3:8, 4:12, 7:15Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷⁻Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹⁻Maa¹²-Xaa¹³-Xaa¹⁴⁻Maa¹⁵-95 XIV Xaa¹⁶ Formula XV 1:6, 2:10, 5:13Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula 1:6, 2:10, 5:13Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³- 97XVI Formula 1:6, 2:10, 5:13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-Xaa_(n2)98 XVII

TABLE III GCRA Peptides Position of Name Disulfide bonds Structure SEQID NO: SP-363 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-99 AMIDE¹⁶ SP-364 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶100 SP-365 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer-101 AMIDE¹⁶ SP-366 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶102 SP-367 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr-103 AMIDE¹⁶ SP-373 C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-104 AMIDE¹⁶ SP-304 di C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶⁻105 PEG PEG3 SP-304 N- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶106 PEG SP-304 C- C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶⁻PEG3107 PEG

TABLE IV SP-304 Analogs, Uroguanylin, and Uroguanylin Analogs Positionof SEQ Name Disulfide bonds Structure ID NO Formula C4:C12,Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa¹⁶108 XVIII C7:C15 Uroguanylin C4:C12,Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶109 C7:C15 N32 C4:C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶110 C7:C15 N33 C4:C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶111 C7:C15 N34 C4:C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶112 C7:C15 N35 C4:C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶113 C7:C15 N36 C4:C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶114 C7:C15 N37 C4:C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶115 C7:C15 N38 C4:C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶116 C7:C15 N39 C4:C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶117 C7:C15 N40 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶118 C7:C15 N41 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶119 C7:C15 N42 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶120 C7:C15 N43 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶121 C7:C15 N44 C4:C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶122 C7:C15 N45 C4:C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶123 C7:C15 N46 C4:C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶124 C7:C15 N47 C4:C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶125 C7:C15 N48 C4:C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶126 C7:C15 N49 C4:C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶127 C7:C15 N50 C4:C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶128 C7:C15 N51 C4:C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶129 C7:C15 N52 C4:C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶130 C7:C15 N53 C4:C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶131 C7:C15 N54 C4:C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶132 C7:C15 N55 C4:C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶133 C7:C15 N56 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶134 C7:C15 N57 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶135 C7:C15 N58 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶136 C7:C15 N59 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶137 C7:C15 N60 C4:C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶138 C7:C15 N61 C4:C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶139 C7:C15 N62 C4:C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶140 C7:C15 N63 C4:C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶141 C7:C15 N65 C4:C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶142 C7:C15 N66 C4:C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶143 C7:C15 N67 C4:C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶144 C7:C15 N68 C4:C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶145 C7:C15 N69 C4:C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶146 C7:C15 N70 C4:C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶147 C7:C15 N71 C4:C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶148 C7:C15 N72 C4:C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶149 C7:C15 N73 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶150 C7:C15 N74 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶151 C7:C15 N75 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶152 C7:C15 N76 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶153 C7:C15 N77 C4:C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶154 C7:C15 N78 C4:C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶155 C7:C15 N79 C4:C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶156 C7:C15 N80 C4:C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶157 C7:C15 N81 C4:C12,Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶158 C7:C15 N82 C4:C12,Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶159 C7:C15 N83 C4:C12,Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶160 C7:C15 N84 C4:C12,Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶161 C7:C15 N85 C4:C12,Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶162 C7:C15 N86 C4:C12,Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶163 C7:C15 N87 C4:C12,Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶164 C7:C15 N88 C4:C12,Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶165 C7:C15 N89 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶166 C7:C15 N90 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶167 C7:C15 N91 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶168 C7:C15 N92 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶169 C7:C15 N93 C4:C12,Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶170 C7:C15 N94 C4:C12,Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶171 C7:C15 N95 C4:C12,Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶172 C7:C15 N96 C4:C12,Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶173 C7:C15

TABLE V Guanylin and Analogs Position of SEQ ID Name Disulfide bondsStructure NO Formula 4:12, 7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵174 XIX Guanylin C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵175 N97 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 N98 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 N99 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 N100 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 N101 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 N102 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 N103 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 N104 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 N105 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 N106 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 N107 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 N108 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 N109 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 N110 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 N111 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 N112 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 N113 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 N114 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 N115 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 N116 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 N117 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 N118 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 N119 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 N120 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 N121 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 N122 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 N123 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 N124 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 N125 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 N126 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 N127 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 N128 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207

TABLE VI Lymphoguanylin and Analogs Position of Disulfide SEQ Name bondsStructure ID NO Formula XX 4:12, 7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 Lymphoguanylin C4:C12Gln¹-Glu²-Glu-³Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵209 N129 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵210 N130 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵211 N131 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵212 N132 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵213 N133 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵214 N134 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵215 N135 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵216 N136 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵217 N137 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵218 N138 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵219 N139 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵220 N140 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵221 N141 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵222 N142 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵223 N143 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵224 N144 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵225 N145 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶226 C7:C15 N146 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶227 C7:C15 N147 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶228 C7:C15 N148 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶229 C7:C15 N149 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶230 C7:C15 N150 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser231 C7:C15 N151 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶232 C7:C15 N152 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶233 C7:C15 N153 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶234 C7:C15 N154 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶235 C7:C15 N155 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶236 C7:C15 N156 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶237 C7:C15 N157 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶238 C7:C15 N158 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶239 C7:C15 N159 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶240 C7:C15 N160 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶241 C7:C15

TABLE VII ST Peptide and Analogues Position of SEQ ID Name Disulfidebonds Structure NO ST C3:C8, C4:C12,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-Cys¹⁴-Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-242 Peptide C7:15 Thr¹⁹-Gly²⁰-Cys²¹-Tyr²² N161 C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3243 C7:15 N162 C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶244 C7:15 N163 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3245 C7:15 N164 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶246 C7:15 N165 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶247 C7:15 N166 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶248 C7:15 N167 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶249 C7:15

1.3 Methods of Use

The invention provides methods for treating or preventinggastrointestinal disorders and increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist formulation to the subject. Non-limiting examples ofgastrointestinal disorders that can be treated or prevented according tothe methods of the invention include irritable bowel syndrome (IBS),non-ulcer dyspepsia, chronic intestinal pseudo-obstruction, functionaldyspepsia, colonic pseudo-obstruction, duodenogastric reflux,gastroesophageal reflux disease (GERD), ileus (e.g., post-operativeileus), gastroparesis, heartburn (high acidity in the GI tract),constipation (e.g., constipation associated with use of medications suchas opioids, osteoarthritis drugs, or osteoporosis drugs); post surgicalconstipation, constipation associated with neuropathic disorders,Crohn's disease, and ulcerative colitis.

In one embodiment, the invention provides methods for treating orpreventing gastrointestinal motility disorder, irritable bowel syndrome,a functional gastrointestinal disorder, gastroesophageal reflux disease,duodenogastric reflux, functional heartburn, dyspepsia, functionaldyspepsia, nonulcer dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction, colonic pseudo-obstruction, obesity, congestiveheart failure, or benign prostatic hyperplasia.

In one embodiment, the invention provides methods for treating orpreventing constipation and/or increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist formulation to the subject. Clinically accepted criteria thatdefine constipation range from the frequency of bowel movements, theconsistency of feces and the ease of bowel movement. One commondefinition of constipation is less than three bowel movements per week.Other definitions include abnormally hard stools or defecation thatrequires excessive straining (Schiller 2001 Aliment Pharmacol Ther15:749-763). Constipation may be idiopathic (functional constipation orslow transit constipation) or secondary to other causes includingneurologic, metabolic or endocrine disorders. These disorders includediabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia,Multiple sclerosis, Parkinson's disease, spinal cord lesions,Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprungdisease and cystic fibrosis. Constipation may also be the result ofsurgery or due to the use of drugs such as analgesics (like opioids),antihypertensives, anticonvulsants, antidepressants, antispasmodics andantipsychotics.

In various embodiments, the constipation is associated with use of atherapeutic agent; the constipation is associated with a neuropathicdisorder; the constipation is postsurgical constipation; theconstipation is associated with a gastrointestinal disorder; theconstipation is idiopathic (functional constipation or slow transitconstipation); the constipation is associated with neuropathic,metabolic or endocrine disorder (e.g., diabetes mellitus,hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis,Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomicneuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis).Constipation may also be the result of surgery or due to the use ofdrugs such as analgesics (e.g., opioids), antihypertensives,anticonvulsants, antidepressants, antispasmodics and antipsychotics.

In one embodiment, the invention provides methods for treating orpreventing chronic idiopathic constipation and increasinggastrointestinal motility in a subject in need thereof by administeringan effective amount of a GCC agonist formulation to the subject.

The term “treating” as used herein refers to a reduction, a partialimprovement, amelioration, or a mitigation of at least one clinicalsymptom associated with the gastrointestinal disorders being treated.The term “preventing” refers to an inhibition or delay in the onset orprogression of at least one clinical symptom associated with thegastrointestinal disorders to be prevented. The term “effective amount”as used herein refers to an amount that provides some improvement orbenefit to the subject. In certain embodiments, an effective amount isan amount that provides some alleviation, mitigation, and/or decrease inat least one clinical symptom of the gastrointestinal disorder to betreated. In other embodiments, the effective amount is the amount thatprovides some inhibition or delay in the onset or progression of atleast one clinical symptom associated with the gastrointestinal disorderto be prevented. The therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. The term“subject” preferably refers to a human subject but may also refer to anon-human primate or other mammal preferably selected from among amouse, a rat, a dog, a cat, a cow, a horse, or a pig.

The invention also provides methods for treating gastrointestinal cancerin a subject in need thereof by administering an effective amount of aGCC agonist formulation to the subject. Non-limiting examples ofgastrointestinal cancers that can be treated according to the methods ofthe invention include gastric cancer, esophageal cancer, pancreaticcancer, colorectal cancer, intestinal cancer, anal cancer, liver cancer,gallbladder cancer, or colon cancer.

The invention also provides methods for treating lipid metabolismdisorders, biliary disorders, inflammatory disorders, lung disorders,cancer, cardiac disorders including cardiovascular disorders, eyedisorders, oral disorders, blood disorders, liver disorders, skindisorders, prostate disorders, endocrine disorders, and obesity.

Lipid metabolism disorders include, but are not limited to,dyslipidemia, hyperlipidemia, hypercholesterolemia,hypertriglyceridemia, sitosterolemia, familial hypercholesterolemia,xanthoma, combined hyperlipidemia, lecithin cholesterol acyltransferasedeficiency, tangier disease, abetalipoproteinemia, erectile dysfunction,fatty liver disease, and hepatitis.

Billary disorders include gallbladder disorders such as for example,gallstones, gall bladder cancer cholangitis, or primary sclerosingcholangitis; or bile duct disorders such as for example, cholecystitis,bile duct cancer or fascioliasis.

Inflammatory disorders include tissue and organ inflammation such askidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); necrotizingenterocolitis (NEC); pancreatic inflammation (e.g., pancreatis), lunginflammation (e.g., bronchitis or asthma) or skin inflammation (e.g.,psoriasis, eczema).

Lung Disorders include for example chronic obstructive pulmonary disease(COPD), and fibrosis.

Cancer includes tissue and organ carcinogenesis including metastasessuch as for example gastrointestinal cancer, (e.g., gastric cancer,esophageal cancer, pancreatic cancer colorectal cancer, intestinalcancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer;lung cancer; thyroid cancer; skin cancer (e.g., melanoma); oral cancer;urinary tract cancer (e.g. bladder cancer or kidney cancer); bloodcancer (e.g. myeloma or leukemia) or prostate cancer.

Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high triglycerides.Cardiovascular disorders include for example aneurysm, angina,atherosclerosis, cerebrovascular accident (stroke),cerebrovasculardisease, congestive heart failure, coronary arterydisease, myocardial infarction (heart attack), or peripheral vasculardisease.

Liver disorders include for example cirrhosis and fibrosis. In addition,GC-C agonist may also be useful to facilitate liver regeneration inliver transplant patients. Eye disorders include for example increasedintra-ocular pressure, glaucoma, dry eyes retinal degeneration,disorders of tear glands or eye inflammation. Skin disorders include forexample xerosis. Oral disorders include for example dry mouth(xerostomia), Sjögren's syndrome, gum diseases (e.g., periodontaldisease), or salivary gland duct blockage or malfunction. Prostatedisorders include for example benign prostatic hyperplasia (BPH).Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

1.3.1 Therapeutically Effective Dosages

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCC agonist peptide. The presentinvention is based in part on the unexpected results of clinical trialsin humans which demonstrated that the formulations of the invention aretherapeutically effective at much lower doses than predicted based onanimal studies. In accordance with one aspect of the invention, therapeutically effective dose is between 0.01 milligrams (mg) and 10 mgper unit dose. The term “unit dose” refers to a single drug deliveryentity, e.g., a tablet, capsule, solution or inhalation formulation. Inone embodiment, the effective dose is between 0.01 mg and 9 mg. Inanother embodiment, the effective dose is between 0.01 mg and 5 mg. Inanother embodiment, the effective dose is between 0.01 mg and 3 mg. Inanother embodiment, the effective dose is between 0.10 mg and 5 mg. Inanother embodiment, the effective dose is between 0.10 mg and 3 mg. Inone embodiment, the unit dose is 0.01 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5 mg, or 10 mg. Inone embodiment, the unit dose is 0.3 mg, 1.0 mg, 3.0 mg, 9.0 mg, or 9.5mg.

The GCC agonist peptides may be in a pharmaceutical composition in unitdose form, together with one or more pharmaceutically acceptableexcipients. The amount of peptide present should be sufficient to have apositive therapeutic effect when administered to a patient. Whatconstitutes a “positive therapeutic effect” will depend upon theparticular condition being treated and will include any significantimprovement in a condition readily recognized by one of skill in theart.

The GCC agonists for use in the methods described above are preferablyadministered orally. Dosage forms include solutions, suspensions,emulsions, tablets, and capsules.

The total daily dose can be administered to the patient in a singledose, or in multiple sub-doses. Typically, sub-doses can be administeredtwo to six times per day, preferably two to four times per day, and evenmore preferably two to three times per day. Preferably, a single dailydose is administered.

The GCC agonists may be administered as either the sole active agent orin combination with one or more additional active agents. In all cases,additional active agents should be administered at a dosage that istherapeutically effective using the existing art as a guide. The GCCagonists may be administered in a single composition or sequentiallywith the one or more additional active agents. In one embodiment, theGCC agonist is administered in combination with one or more inhibitorsof cGMP dependent phosphodiesterase such as suldinac sulfone, zaprinast,motapizone, vardenafil, or sildenifil. In another embodiment, the GCCagonist is administered in combination with one or more chemotherapeuticagents. In another embodiment, the GCC agonist is administered incombination with one or more or anti-inflammatory drugs such as steroidsor non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin.

Combination therapy can be achieved by administering two or more agents,e.g., a GCC agonist peptide described herein and another compound, eachof which is formulated and administered separately, or by administeringtwo or more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination therapy can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, days, or weeks. Thus, the two or more agentscan be administered within minutes of each other or within 1, 2, 3, 6,9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or10 weeks of each other. In some cases even longer intervals arepossible. While in many cases it is desirable that the two or moreagents used in a combination therapy be present in within the patient'sbody at the same time, this need not be so.

The GCC agonist peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

1.3.2 Exemplary Agents for Combination Therapy

The GCC agonist formulations of the invention may be administered aloneor in combination with one or more additional therapeutic agents as partof a therapeutic regimen for the treatment or prevention of agastrointestinal disease or disorder. In some embodiments, the GCCagonist formulation comprises one or more additional therapeutic agents.In other embodiments, the GCC agonist is formulated separately from theone or more additional therapeutic agents. In accordance with thisembodiment, the GCC agonist is administered either simultaneously,sequentially, or at a different time than the one or more additionaltherapeutic agents. In one embodiment, the GCC agonist formulation isadministered in combination with one or more additional therapeuticagents selected from the group consisting of phosphodiesteraseinhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative (suchas SENNA or METAMUCIL), a stool softner, an anti-tumor necrosis factoralpha therapy for IBD (such as REMICADE, ENBREL, or HUMIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters. Further examples of therapeutic agents that maybe administered in combination with the GCC agonist formulations of theinvention are given in the following sections.

1.3.2.1 Agents to Treat Gastrointestinal Cancers

The GCC agonist formulations described herein can be used in combinationwith one or more antitumor agents including but not limited toalkylating agents, epipodophyllotoxins, nitrosoureas, anti-metabolites,vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, andthe like. Particular antitumor agents include tamoxifen, taxol,etoposide, and 5-fluorouracil. In one embodiment, the GCC agonistformulations are used in combination with an antiviral agent or amonoclonal antibody.

Non-limiting examples of antitumor agents that can be used incombination with the GCC agonist formulations of the invention for thetreatment of colon cancer include anti-proliferative agents, agents forDNA modification or repair, DNA synthesis inhibitors, DNA/RNAtranscription regulators, RNA processing inhibitors, agents that affectprotein expression, synthesis and stability, agents that affect proteinlocalization or their ability to exert their physiological action,agents that interfere with protein-protein or protein-nucleic acidinteractions, agents that act by RNA interference, receptor bindingmolecules of any chemical nature (including small molecules andantibodies), targeted toxins, enzyme activators, enzyme inhibitors, generegulators, HSP-90 inhibitors, molecules interfering with microtubulesor other cytoskeletal components or cell adhesion and motility, agentsfor phototherapy, and therapy adjuncts.

Representative anti-proliferative agents include N-acetyl-D-sphingosine(C.sub.2 ceramide), apigenin, berberine chloride,dichloromethylenediphosphonic acid disodium salt, loe-emodine, emodin,HA 14-1, N-hexanoyl-D-sphingosine (C.sub.6 ceramide),7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin, parthenolide,and rapamycin.

Representative agents for DNA modification and repair includeaphidicolin, bleomycin sulfate, carboplatin, carmustine, chlorambucil,cyclophosphamide monohydrate, cyclophosphamide monohydrate ISOPAC®,cis-diammineplatinum(II) dichloride (Cisplatin), esculetin, melphalan,methoxyamine hydrochloride, mitomycin C, mitoxantrone dihydrochloride,oxaliplatin, and streptozocin.

Representative DNA synthesis inhibitors include (.+−.)amethopterin(methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine b-D-arabinofurdnoside (Ara-C), cytosine b-D-arabinofuranoside(Ara-C) hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside(Fludarabine des-phosphate; F-ara-A), 5-fluoro-5′-deoxyuridinc,5-fluorouracil, ganciclovir, hydroxyurea, 6-mercaptopurine, and6-thioguanine.

Representative DNA/RNA transcription regulators include actinomycin D,daunorubicin hydrochloride, 5,6-dichlorobenzimidazole1-b-D-ribofuranoside, doxorubicin hydrochloride, homoharringtonine, andidarubicin hydrochloride.

Representative enzyme activators and inhibitors include forskolin,DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein,(S)-(+)-camptothecin, curcumin, (−)-deguelin, (−)-depudecin, doxycyclinehyclate, etoposide, formestane, fostriecin sodium salt, hispidin,2-imino-1-imidazolidineacetic acid (Cyclocreatine), oxamflatin,4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin A,tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment,valproic acid (2-propylpentanoic acid), and XK469.

Representative gene regulators include 5-aza-2′-deoxycytidine,5-azacytidine, cholecalciferol (Vitamin D3), ciglitizone, cyproteroneacetate, 15-deoxy-D.sup.12,14-prostaglandin J.sub.2, epitestosterone,flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin),4-hydroxytamoxifen, mifepri stone, procainamide hydrochloride,raloxifene hydrochloride, all trans-retinal (vitamin A aldehyde),retinoic acid (vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoicacid, retinoic acid p-hydroxyanilide, retinol (Vitamin A), tamoxifen,tamoxifen citrate salt, tetradecylthioacetic acid, and troglitazone.

Representative HSP-90 inhibitors include17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.

Representative microtubule inhibitors include colchicines, dolastatin15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin,rhizoxin, vinblastine sulfate salt, vincristine sulfate salt, andvindesine sulfate salt and vinorelbine (Navelbine) ditartrate salt.

Representative agents for performing phototherapy include photoactiveporphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen,psoralen and ursodeoxycholic acid.

Representative agents used as therapy adjuncts include amifostine,4-amino-1,8-naphthalimide, brefeldin A, cimetidine, phosphomycindisodium salt, leuprolide (leuprorelin) acetate salt, luteinizinghormone-releasing hormone (LH-RH) acetate salt, lectin, papaverinehydrochloride, pifithrin-a, (−)-scopolamine hydrobromide, andthapsigargin.

The agents can also be anti-VEGF (vascular endothelial growth factor)agents, as such are known in the art. Several antibodies and smallmolecules are currently in clinical trials or have been approved thatfunction by inhibiting VEGF, such as Avastin (Bevacizumab), SU5416,SU11248 and BAY 43-9006. The agents can also be directed against growthfactor receptors such as those of the EGF/Erb-B family such as EGFReceptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2,receptor (Herceptin or Trastuzumab), other receptors (such as Rituximabor Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases,cellular serine/threonine kinases (including MAP kinases), and variousother proteins whose deregulation contribute to oncogenesis (such assmall/Ras family and large/heterotrimeric G proteins). Severalantibodies and small molecules targeting those molecules are currentlyat various stages of development (including approved for treatment or inclinical trials).

In a preferred embodiment, the invention provides a method for treatingcolon cancer in a subject in need thereof by administering to thesubject a GCC agonist formulation in combination with one or moreantitumor agent selected from the group consisting of paclitaxel,docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin, etoposide,topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine,cyclophosphamide, gentuzumab, carboplatin, interferons, and doxorubicin.In a particular embodiment the antitumor agent is paclitaxel. In afurther embodiment, the method further comprises an antitumor agentselected from the group consisting of 5-FU, doxorubicin, vinorelbine,cytoxan, and cisplatin.

1.3.2.2 Agents that Treat Crohn's Disease

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of Crohn's disease.Non-limiting examples of the one or more additional therapeutic agentsinclude sulfasalazine and other mesalamine-containing drugs, generallyknown as 5-ASA agents, such as Asacol, Dipentum, or Pentasa, orinfliximab (REMICADE). In certain embodiments, the one or moreadditional agents is a corticosteroid or an immunosuppressive agent suchas 6-mercaptopurine or azathioprine. In another embodiment, the one ormore additional agents is an antidiarrheal agent such as diphenoxylate,loperamide, or codeine.

1.3.2.3 Agents that Treat Ulcerative Colitis

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of ulcerative colitis.The agents that are used to treat ulcerative colitis overlap with thoseused to treat Chrohn's Disease. Non-limiting examples of the one or moreadditional therapeutic agents that can be used in combination with a GCCagonist formulation of the invention include aminosalicylates (drugsthat contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine,olsalazine, mesalamine, and balsalazide. Other therapeutic agents thatcan be used include corticosteroids, such as prednisone andhydrocortisone, immunomodulators, such as azathioprine,6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines, andanti-TNF-alpha agents, including the thiazolidinediones or glitazonessuch as rosiglitazone and pioglitazone. In one emobidment, the one ormore additional therapeutic agents includes both cyclosporine A and 6-MPor azathioprine for the treatment of active, severe ulcerative colitis.

1.3.2.4 Agents that Treat Constipation/Irritable Bowel Syndrome

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of constipation, such asthat associated with irritable bowel syndrome. Non-limiting examples ofthe one or more additional therapeutic agents include laxatives such asSENNA, MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), stoolsofteners (such as mineral oil or COLACE), bulking agents (such asMETAMUCIL or bran), agents such as ZELNORM (also called tegaserod), andanticholinergic medications such as BENTYL and LEVSIN.

1.3.2.5 Agents for the Treatment of Postoperative Ileus

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of postoperative ileus.Non-limiting examples of the one or more additional therapeutic agentsinclude ENTEREG (alvimopan; formerly called ado lor/ADL 8-2698),conivaptan, and related agents describes in U.S. Pat. No. 6,645,959.

1.3.2.6 Anti-Obesity Agents

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of obesity. Non-limitingexamples of the one or more additional therapeutic agents include 110HSD-I (11-beta hydroxy steroid dehydrogenase type 1) inhibitors, such asBVT 3498, BVT 2733,3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]annulene,and those compounds disclosed in WO01/90091, WOO 1/90090, WOO 1/90092and WO02/072084; 5HT antagonists such as those in WO03/037871,WO03/037887, and the like; 5HTIa modulators such as carbidopa,benserazide and those disclosed in U.S. Pat. No. 6,207,699, WO03/031439,and the like; 5HT2c (serotonin receptor 2c) agonists, such as BVT933,DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (Glaxo SmithKline) and YM 348 and those disclosed in U.S. Pat. No. 3,914,250,WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548, WO02/44152,WO02/51844, WO02/40456, and WO02/40457; 5HT6 receptor modulators, suchas those in WO03/030901, WO03/035061, WO03/039547, and the like;acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M.et al, Obesity Research, 9:202-9 (2001) and Japanese Patent ApplicationNo. JP 2000256190; anorectic bicyclic compounds such as 1426 (Aventis)and 1954 (Aventis), and the compounds disclosed in WO00/18749,WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB 1 (cannabinoid-1receptor) antagonist/inverse agonists such as rimonabant (Acomplia;Sanofi), SR-147778 (Sanofi), SR-141716 (Sanofi), BAY 65-2520 (Bayer),and SLV 319 (Solvay), and those disclosed in patent publications U.S.Pat. No. 4,973,587, U.S. Pat. No. 5,013,837, U.S. Pat. No. 5,081,122,U.S. Pat. No. 5,112,820, U.S. Pat. No. 5,292,736, U.S. Pat. No.5,532,237, U.S. Pat. No. 5,624,941, U.S. Pat. No. 6,028,084, U.S. Pat.No. 6,509,367, U.S. Pat. No. 6,509,367, WO96/33159, WO97/29079,WO98/31227, WO98/33765, WO98/37061, WO98/41519, WO98/43635, WO98/43636,WO99/02499, WO00/10967, WO00/10968, WO01/09120, WO01/58869, WO01/64632,WO01/64633, WO01/64634, WO01/70700, WO01/96330, WO02/076949,WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648,WO03/027069, WO03/027076, WO03/027114, WO03/037332, WO03/040107,WO03/086940, WO03/084943 and EP658546; CCK-A (cholecystokinin-A)agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378, A-71623and SR146131 (Sanofi), and those described in U.S. Pat. No. 5,739,106;CNTF (Ciliary neurotrophic factors), such as GI-181771(Glaxo-SmithKline), SRI 46131 (Sanofi Synthelabo), butabindide, PD170,292, and PD 149164 (Pfizer); CNTF derivatives, such as Axokine®(Regeneron), and those disclosed in WO94/09134, WO98/22128, andWO99/43813; dipeptidyl peptidase IV (DP-IV) inhibitors, such asisoleucine thiazolidide, valine pyrrolidide, NVP-DPP728, LAF237, P93/01,P 3298, TSL 225 (tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid; disclosed by Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998)1537-1540), TMC-2A/2B/2 C, CD26 inhibtors, FE 999011, P9310/1(364, VIP0177, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996) and the compounds disclosed patentpublications. WO99/38501, WO99/46272, WO99/67279 (Probiodrug),WO99/67278 (Probiodrug), WO99/61431 (Probiodrug), WO02/083128,WO02/062764, WO03/000180, WO03/000181, WO03/000250, WO03/002530,WO03/002531, WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936, WO03/024942, WO03/024965, WO03/033524, WO03/037327 andEP1258476; growth hormone secretagogue receptor agonists/antagonists,such as NN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391(Pfizer), LY 444,711 (Eli Lilly), L-692,429 and L-163,255, and such asthose disclosed in U.S. Ser. No. 09/662,448, U.S. provisionalapplication 60/203,335, U.S. Pat. No. 6,358,951, US2002049196,US2002/022637, WO01/56592 and WO02/32888; H3 (histamine H3)antagonist/inverse agonists, such as thioperamide,3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit,iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440,O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al.,Pharmazie, 55:349-55 (2000)), piperidine-containing histamineH3-receptor antagonists (Lazewska, D. et al., Pharmazie, 56:927-32(2001), benzophenone derivatives and related compounds (Sasse, A. etal., Arch. Pharm. (Weinheim) 334:45-52 (2001)), substitutedN-phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)),and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43(2000)) and histamine H3 receptor modulators such as those disclosed inWO02/15905, WO03/024928 and WO03/024929; leptin derivatives, such asthose disclosed in U.S. Pat. No. 5,552,524, U.S. Pat. No. 5,552,523,U.S. Pat. No. 5,552,522, U.S. Pat. No. 5,521,283, WO96/23513,WO96/23514, WO96/23515, WO96/23516, WO96/23517, WO96/23518, WO96/23519,and WO96/23520; leptin, including recombinant human leptin (PEG-OB,Hoffman La Roche) and recombinant methionyl human leptin (Amgen); lipaseinhibitors, such as tetrahydrolipstatin (orlistat/Xenical®), Triton WRl339, RHC80267, lipstatin, teasaponin, diethylumbelliferyl phosphate,FL-386, WAY-121898, Bay-N-3176, valilactone, esteracin, ebelactone A,ebelactone B, and RHC 80267, and those disclosed in patent publicationsWO01/77094, U.S. Pat. No. 4,598,089, U.S. Pat. No. 4,452,813,USUS5512565, U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151, U.S. Pat.No. 4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No. 4,242,453;lipid metabolism modulators such as maslinic acid, erythrodiol, ursolicacid uvaol, betulinic acid, betulin, and the like and compoundsdisclosed in WO03/011267; Mc4r (melanocortin 4 receptor) agonists, suchas CHIR86036 (Chiron), ME-10142, ME-10145, and HS-131 (Melacure), andthose disclosed in PCT publication Nos. WO99/64002, WO00/74679, WOO1/991752, WOO 1/25192, WOO 1/52880, WOO 1/74844, WOO 1/70708,WO01/70337, WO01/91752, WO02/059095, WO02/059107, WO02/059108,WO02/059117, WO02/06276, WO02/12166, WO02/11715, WO02/12178, WO02/15909,WO02/38544, WO02/068387, WO02/068388, WO02/067869, WO02/081430,WO03/06604, WO03/007949, WO03/009847, WO03/009850, WO03/013509, andWO03/031410; Mc5r (melanocortin 5 receptor) modulators, such as thosedisclosed in WO97/19952, WO00/15826, WO00/15790, US20030092041;melanin-concentrating hormone 1 receptor (MCHR) antagonists, such asT-226296 (Takeda), SB 568849, SNP-7941 (Synaptic), and those disclosedin patent publications WOO 1/21169, WO01/82925, WO01/87834, WO02/051809,WO02/06245, WO02/076929, WO02/076947, WO02/04433, WO02/51809,WO02/083134, WO02/094799, WO03/004027, WO03/13574, WO03/15769,WO03/028641, WO03/035624, WO03/033476, WO03/033480, JP13226269, andJP1437059; mGluR5 modulators such as those disclosed in WO03/029210,WO03/047581, WO03/048137, WO03/051315, WO03/051833, WO03/053922,WO03/059904, and the like; serotoninergic agents, such as fenfluramine(such as Pondimin® (Benzeneethanamine,N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Robbins),dexfenfluramine (such as Redux® (Benzeneethanamine,N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Interneuron)and sibutramine ((Meridia®, Knoll/Reductil™) including racemic mixtures,as optically pure isomers (+) and (−), and pharmaceutically acceptablesalts, solvents, hydrates, clathrates and prodrugs thereof includingsibutramine hydrochloride monohydrate salts thereof, and those compoundsdisclosed in U.S. Pat. No. 4,746,680, U.S. Pat. No. 4,806,570, and U.S.Pat. No. 5,436,272, US20020006964, WOO 1/27068, and WOO 1/62341; NE(norepinephrine) transport inhibitors, such as GW 320659, despiramine,talsupram, and nomifensine; NPY 1 antagonists, such as BIBP3226,J-115814, BIBO 3304, LY-357897, CP-671906, GI-264879A, and thosedisclosed in U.S. Pat. No. 6,001,836, WO96/14307, WO01/23387,WO99/51600, WO01/85690, WO01/85098, WO01/85173, and WO01/89528; NPYS(neuropeptide Y Y5) antagonists, such as 152,804, GW-569180A,GW-594884A, GW-587081X, GW-548118X, FR235208, FR226928, FR240662,FR252384, 1229U91, GI-264879A, CGP71683A, LY-377897, LY-366377,PD-160170, SR-120562A, SR-120819A, JCF-104, and H409/22 and thosecompounds disclosed in patent publications U.S. Pat. No. 6,140,354, U.S.Pat. No. 6,191,160, U.S. Pat. No. 6,218,408, U.S. Pat. No. 6,258,837,U.S. Pat. No. 6,313,298, U.S. Pat. No. 6,326,375, U.S. Pat. No.6,329,395, U.S. Pat. No. 6,335,345, U.S. Pat. No. 6,337,332, U.S. Pat.No. 6,329,395, U.S. Pat. No. 6,340,683, EP01010691, EP-01044970,WO97/19682, WO97/20820, WO97/20821, WO97/20822, WO97/20823, WO98/27063,WO00/107409, WO00/185714, WO00/185730, WO00/64880, WO00/68197,WO00/69849, WO/0113917, WO01/09120, WO01/14376, WO01/85714, WO01/85730,WO01/07409, WO01/02379, WO01/23388, WO01/23389, WOO 1/44201, WO01/62737,WO01/62738, WO01/09120, WO02/20488, WO02/22592, WO02/48152, WO02/49648,WO02/051806, WO02/094789, WO03/009845, WO03/014083, WO03/022849,WO03/028726 and Norman et al, J. Med. Chem. 43:4288-4312 (2000); opioidantagonists, such as nalmefene (REVEX®), 3-methoxynaltrexone,methylnaltrexone, naloxone, and naltrexone (e.g. PT901; PainTherapeutics, Inc.) and those disclosed in US20050004155 and WO00/21509;orexin antagonists, such as SB-334867-A and those disclosed in patentpublications WO01/96302, WO01/68609, WO02/44172, WO02/51232, WO02/51838,WO02/089800, WO02/090355, WO03/023561, WO03/032991, and WO03/037847; PDEinhibitors (e.g. compounds which slow the degradation of cyclic AMP(cAMP) and/or cyclic GMP (cGMP) by inhibition of the phosphodiesterases,which can lead to a relative increase in the intracellular concentrationof cAMP and cGMP; possible PDE inhibitors are primarily those substanceswhich are to be numbered among the class consisting of the PDE3inhibitors, the class consisting of the PDE4 inhibitors and/or the classconsisting of the PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors) such as those disclosed in patent publicationsDE1470341, DE2108438, DE2123328, DE2305339, DE2305575, DE2315801,DE2402908, DE2413935, DE2451417, DE2459090, DE2646469, DE2727481,DE2825048, DE2837161, DE2845220, DE2847621, DE2934747, DE3021792,DE3038166, DE3044568, EP000718, EP0008408, EP0010759, EP0059948,EP0075436, EP0096517, EPO1 12987, EPO1 16948, EP0150937, EP0158380,EP0161632, EP0161918, EP0167121, EP0199127, EP0220044, EP0247725,EP0258191, EP0272910, EP0272914, EP0294647, EP0300726, EP0335386,EP0357788, EP0389282, EP0406958, EP0426180, EP0428302, EP0435811,EP0470805, EP0482208, EP0490823, EP0506194, EP0511865, EP0527117,EP0626939, EP0664289, EP0671389, EP0685474, EP0685475, EP0685479,JP92234389, JP94329652, JP95010875, U.S. Pat. No. 4,963,561, U.S. Pat.No. 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146, WO9315044,WO9315045, WO9318024, WO9319068, WO9319720, WO9319747, WO9319749,WO9319751, WO9325517, WO9402465, WO9406423, WO9412461, WO9420455,WO9422852, WO9425437, WO9427947, WO9500516, WO9501980, WO9503794,WO9504045, WO9504046, WO9505386, WO9508534, WO9509623, WO9509624,WO9509627, WO9509836, WO9514667, WO9514680, WO9514681, WO9517392,WO9517399, WO9519362, WO9522520, WO9524381, WO9527692, WO9528926,WO9535281, WO9535282, WO9600218, WO9601825, WO9602541, WO9611917,DE3142982, DEl 116676, DE2162096, EP0293063, EP0463756, EP0482208,EP0579496, EP0667345 U.S. Pat. No. 6,331,543, US20050004222 (includingthose disclosed in formulas I-XIII and paragraphs 37-39, 85-0545 and557-577), WO9307124, EP0163965, EP0393500, EP0510562, EP0553174,WO9501338 and WO9603399, as well as PDE5 inhibitors (such as RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra™)), PDE4 inhibitors (such asetazolate, ICI63197, RP73401, imazolidinone (RO-20-1724), MEM 1414(R1533/R1500; Pharmacia Roche), denbufylline, rolipram, oxagrelate,nitraquazone, Y-590, DH-6471, SKF-94120, motapizone, lixazinone,indolidan, olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351,atizoram, arofylline, filaminast, PDB-093, UCB-29646, CDP-840,SKF-107806, piclamilast, RS-17597, RS-25344-000, SB-207499, TIBENELAST,SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840,mopidamol, anagrelide, ibudilast, amrinone, pimobendan, cilostazol,quazinone andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide,PDE3 inhibitors (such as ICI153, 100, bemorandane (RWJ 22867), MCI-154,UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran, piroximone,imazodan, CI-930, siguazodan, adibendan, saterinone, SKF-95654,SDZ-MKS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307,revizinone, NM-702, WIN-62582 and WIN-63291, enoximone and milrinone,PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG-30029,zardaverine, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andtolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine,enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast,tadalafil(Cialis®), theophylline, and vardenafil(Levitra®); NeuropeptideY2 (NPY2) agonists include but are not limited to: polypeptide YY andfragments and variants thereof (e.g. YY3-36 (PYY3-36)(N. Engl. J. Med.349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO:XXX))and PYY agonists such as those disclosed in WO02/47712, WO03/026591,WO03/057235, and WO03/027637; serotonin reuptake inhibitors, such as,paroxetine, fluoxetine (Prozac™), fluvoxamine, sertraline, citalopram,and imipramine, and those disclosed in U.S. Pat. No. 6,162,805, U.S.Pat. No. 6,365,633, WO03/00663, WOO 1/27060, and WOO 1/162341; thyroidhormone β agonists, such as KB-2611 (KaroBioBMS), and those disclosed inWO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S. ProvisionalApplication No. 60/183,223, and Japanese Patent Application No. JP2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators, such asphytanic acid, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoicacid (TTNPB), retinoic acid, and those disclosed in WO99/00123; β3 (betaadrenergic receptor 3) agonists, such as AJ9677/TAK677(Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243,GW 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604(Lilly), SR 59119A, and those disclosed in U.S. Pat. No. 5,541,204, U.S.Pat. No. 5,770,615, U.S. Pat. No. 5,491,134, U.S. Pat. No. 5,776,983,US488064, U.S. Pat. No. 5,705,515, U.S. Pat. No. 5,451,677, WO94/18161,WO95/29159, WO97/46556, WO98/04526 and WO98/32753, WO01/74782,WO02/32897, WO03/014113, WO03/016276, WO03/016307, WO03/024948,WO03/024953 and WO03/037881; noradrenergic agents including, but notlimited to, diethylpropion (such as Tenuate® (1-propanone,2-(diethylamino)-1-phenyl-, hydrochloride), Merrell), dextroamphetamine(also known as dextroamphetamine sulfate, dexamphetamine, dexedrine,Dexampex, Ferndex, Oxydess II, Robese, Spancap #1), mazindol ((or5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-01) such asSanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (orBenzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((orPhenol, 3-[[4,5-duhydro-1H-imidazol-2-yl)ethyl](4-methylpheny-1)amino],monohydrochloride) such as Adipex-P®, Lemmon, FASTIN®, SmithKlineBeecham and Ionamin®, Medeva), phendimetrazine ((or(2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) such asMetra® (Forest), Plegine® (Wyeth-Ay erst), Prelu-2® (BoehringerIngelheim), and Statobex® (Lemmon), phendamine tartrate (such asThephorin®(2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridineL-(+)-tartrate (1:1)), Hoffmann-LaRoche), methamphetamine (such asDesoxyn®, Abbot ((S)-N, (alpha)-dimethylbenzeneethanaminehydrochloride)), and phendimetrazine tartrate (such as Bontril®Slow-Release Capsules, Amarin (-3,4-Dimethyl-2-phenylmorpholineTartrate); fatty acid oxidation upregulator/inducers such as Famoxin®(Genset); monamine oxidase inhibitors including but not limited tobefloxatone, moclobemide, brofaromine, phenoxathine, esuprone, befol,toloxatone, pirlindol, amiflamine, sercloremine, bazinaprine,lazabemide, milacemide, caroxazone and other certain compounds asdisclosed by WO01/12176; and other anti-obesity agents such as 5HT-2agonists, ACC (acetyl-CoA carboxylase) inhibitors such as thosedescribed in WO03/072197, alpha-lipoic acid (alpha-LA), AOD9604,appetite suppressants such as those in WO03/40107, ATL-962 (AlizymePLC), benzocaine, benzphetamine hydrochloride (Didrex), bladderwrack(focus vesiculosus), BRS3 (bombesin receptor subtype 3) agonists,bupropion, caffeine, CCK agonists, chitosan, chromium, conjugatedlinoleic acid, corticotropin-releasing hormone agonists,dehydroepiandrosterone, DGAT1 (diacylglycerol acyltransferase 1)inhibitors, DGAT2 (diacylglycerol acyltransferase 2) inhibitors,dicarboxylate transporter inhibitors, ephedra, exendin-4 (an inhibitorof glp-1) FAS (fatty acid synthase) inhibitors (such as Cerulenin andC75), fat resorption inhibitors (such as those in WO03/053451, and thelike), fatty acid transporter inhibitors, natural water soluble fibers(such as psyllium, plantago, guar, oat, pectin), galanin antagonists,galega (Goat's Rue, French Lilac), garcinia cambogia, germander(teucrium chamaedrys), ghrelin antibodies and ghrelin antagonists (suchas those disclosed in WO01/87335, and WO02/08250), polypeptide hormonesand variants thereof which affect the islet cell secretion, such as thehormones of the secretin/gastric inhibitory polypeptide (GIP)/vasoactiveintestinal polypeptide (VIP)/pituitary adenylate cyclase activatingpolypeptide (PACAP)/glucagon-like polypeptide II(GLP-II)/glicentin/glucagon gene family and/or those of theadrenomedullin/amylin/calcitonin gene related polypeptide (CGRP) genefamily includingGLP-1 (glucagon-like polypeptide 1) agonists (e.g. (1)exendin-4, (2) those GLP-I molecules described in US20050130891including GLP-1(7-34), GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in itsC-terminally carboxylated or amidated form or as modified GLP-Ipolypeptides and modifications thereof including those described inparagraphs 17-44 of US20050130891, and derivatives derived fromGLP-1-(7-34)COOH and the corresponding acid amide are employed whichhave the following general formula:R—NH-HAEGTFTSDVSYLEGQAAKEFIAWLVK-CONH2 wherein R═H or an organiccompound having from 1 to 10 carbon atoms. Preferably, R is the residueof a carboxylic acid. Particularly preferred are the followingcarboxylic acid residues: formyl, acetyl, propionyl, isopropionyl,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl.) andglp-1 (glucagon-like polypeptide-1), glucocorticoid antagonists, glucosetransporter inhibitors, growth hormone secretagogues (such as thosedisclosed and specifically described in U.S. Pat. No. 5,536,716),interleukin-6 (IL-6) and modulators thereof (as in WO03/057237, and thelike), L-carnitine, Mc3r (melanocortin 3 receptor) agonists, MCH2R(melanin concentrating hormone 2R) agonist/antagonists, melaninconcentrating hormone antagonists, melanocortin agonists (such asMelanotan II or those described in WO 99/64002 and WO 00/74679), nomameherba, phosphate transporter inhibitors, phytopharm compound 57 (CP644,673), pyruvate, SCD-I (stearoyl-CoA desaturase-1) inhibitors, T71(Tularik, Inc., Boulder Colo.), Topiramate (Topimax®, indicated as ananti-convulsant which has been shown to increase weight loss),transcription factor modulators (such as those disclosed inWO03/026576), β-hydroxy steroid dehydrogenase-1 inhibitors (β-HSD-I),β-hydroxy-β-methylbutyrate, p57 (Pfizer), Zonisamide (Zonegran™,indicated as an anti-epileptic which has been shown to lead to weightloss), and the agents disclosed in US20030119428 paragraphs 20-26.

1.3.2.7 Phosphodiesterase Inhibitors

In certain embodiments, the regimen of combination therapy includes theadministration of one or more phosphodiesterase (“PDE”) inhibitors. PDEinhibitors slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP(cGMP) by inhibiting phosphodiesterases, which can lead to a relativeincrease in the intracellular concentration of cAMP and/or cGMP.Non-limiting examples of PDE inhibitors that can be used in combinationwith the GCC agonists of the invention include PDE3 inhibitors, PDE4inhibitors and/or PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors. Non-limiting examples of such PDE inhibitors aredescribed in the following patent applications and patents: DE1470341,DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908,DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048,DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166,DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436,EP0096517, EPO1 12987, EPO1 16948, EP0150937, EP0158380, EP0161632,EP0161918, EP0167121, EP0199127, EP0220044, EP0247725, EP0258191,EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788,EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939,EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389,JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991,WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024,WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517,WO9402465, WO9406423, WO9412461, WO9420455, WO9422852, WO9425437,WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046,WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836,WO9514667, WO9514680, WO9514681, WO9517392, WO9517399, WO9519362,WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282,WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DEl 116676,DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345 U.S.Pat. No. 6,331,543, US20050004222 (including those disclosed in formulasI—XIII and paragraphs 37-39, 85-0545 and 557-577) and WO9307124,EP0163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399.PDE5 inhibitors which may be mentioned by way of example are RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra®). PDE4 inhibitors which may bementioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500;Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE,Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE,ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE,FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL,ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil(Cialis®), theophylline, and vardenafil(Levitra®),zaprinast (PDE5 specific). GCC AGONIST

1.3.2.8 Analgesic Agents

In certain embodiments, the regimen of combination therapy includes theadministration of one or more analgesic agents, e.g., an analgesiccompound or an analgesic polypeptide. In some embodiments, the GCCagonist formulation is administered simultaneously or sequentially withone or more analgesic agents. In other embodiments, the GCC agonist iscovalently linked or attached to an analgesic agent to create atherapeutic conjugate. Non-limiting examples of analgesic agents thatcan be used include calcium channel blockers, 5HT receptor antagonists(for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSRI), vanilloid and cannabanoid receptor agonists, andsialorphin. Further examples of analgesic agents in the various classesare known in the art.

In one embodiment, the analgesic agent is an analgesic polypeptideselected from the group consisting of sialorphin-related polypeptides,including those comprising the amino acid sequence QHNPR (SEQ ID NO:239), including: VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: 241);VRGQHNPR (SEQ ID NO: 242); VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQID NO: 244); VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246).Sialorphin-related polypeptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or polypeptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the GCC agonistsdescribed herein or covalently linked to a GCC agonist to form atherapeutic conjugate. Sialorphin and related polypeptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

In another embodiment, a GCC agonist formulation of the invention isadministered as part of a regimen of combination therapy with an opioidreceptor antagonist or agonist. In one embodiment, the GCC agonist andthe opioid receptor antagonist or agonist are linked via a covalentbond. Non-limiting examples of opioid receptor antagonists includenaloxone, naltrexone, methyl nalozone, nalmefene, cypridime, betafunaltrexamine, naloxonazine, naltrindole, nor-binaltorphimine,enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine),trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons.Non-limiting examples of opioid receptor agonists include fedotozine,asimadoline, and ketocyclazocine, the compounds described in WO03/097051and WO05/007626, morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH 2; WO 01/019849 A1), and loperamide.

Further non-limiting examples of analgesic agents that can be used in aregimen of combination therapy along with the GCC agonist formulationsof the invention include the dipeptide Tyr-Arg (kyotorphin); thechromogranin-derived polypeptide (CgA 47-66; See, e.g., Ghia et al. 2004Regulatory polypeptides 119:199); CCK receptor agonists such ascaerulein; conotoxin polypeptides; peptide analogs of thymulin (FRApplication 2830451); CCK (CCKa or CCKb) receptor antagonists, includingloxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride,metoclopramide, zacopride, cisapride, renzapride, benzimidazolonederivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium channelblockers such as ziconotide and related compounds described in, forexample, EP625162B1, U.S. Pat. No. 5,364,842, U.S. Pat. No. 5,587,454,U.S. Pat. No. 5,824,645, U.S. Pat. No. 5,859,186, U.S. Pat. No.5,994,305, U.S. Pat. No. 6,087,091, U.S. Pat. No. 6,136,786, WO 93/13128A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, U.S. Pat. No. 5,795,864,U.S. Pat. No. 5,891,849, U.S. Pat. No. 6,054,429, WO 97/01351 A1; NK-I,receptor antagonists such as aprepitant (Merck & Co Inc), vofopitant,ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd), SR-48968(Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (Glaxo SmithKline), TAK-637 (Takeda/Abbot), SR-14033, and related compoundsdescribed in, for example, EP 873753 A1, US 20010006972 A1, US20030109417 A1, WO 01/52844 A1 (for a review see Giardina et al. 2003.Drugs 6:758); NK-2 receptor antagonists such as nepadutant (MenariniRicerche SpA), saredutant (Sanoft-Synthelabo), GW597599 (Glaxo SmithKline), SR-144190 (Sanoft-Synthelabo) and UK-290795 (Pfizer Inc); NK3receptor antagonists such as osanetant (SR-142801; Sanoft-Synthelabo),SSR-241586, talnetant and related compounds described in, for example,WO 02/094187 A2, EP 876347 A1, WO 97/21680 A1, U.S. Pat. No. 6,277,862,WO 98/1 1090, WO 95/28418, WO 97/19927, and Boden et al. (J Med Chem.39:1664-75, 1996); norepinephrine-serotonin reuptake inhibitors (NSRI)such as milnacipran and related compounds described in WO 03/077897; andvanilloid receptor antagonists such as arvanil and related compoudsdescribed in WO 01/64212 A1.

In addition to sialorphin-related polypeptides, analgesic polypeptidesinclude: AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin,lupron, ziconotide, and substance P.

1.3.2.9 Insulin and Insulin Modulating Agents

The GCC agonist peptides described herein can be used in combinationtherapy with insulin and related compounds including primate, rodent, orrabbit insulin including biologically active variants thereof includingallelic variants, more preferably human insulin available in recombinantform. Sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin). See,the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins).

The GCC peptides described herein can also be used in combinationtherapy with agents that can boost insulin effects or levels of asubject upon administration, e.g. glipizide and/or rosiglitazone. Thepolypeptides and agonistsdescribed herein can be used in combitherapywith SYMLIN® (pramlintide acetate) and Exenatide® (synthetic exendin-4;a 39 aa polypeptide).

1.3.2.10 Anti-Hypertensive Agents

The GCC agonist peptides described herein can be used in combinationtherapy with an anti-hypertensive agent including but not limited to:(1) diuretics, such as thiazides, including chlorthalidone,chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide,polythiazide, and hydrochlorothiazide; loop diuretics, such asbumetanide, ethacrynic acid, furosemide, and torsemide; potassiumsparing agents, such as amiloride, and triamterene; carbonic anhydraseinhibitors, osmotics (such as glycerin) and aldosterone antagonists,such as spironolactone, epirenone, and the like; (2) beta-adrenergicblockers such as acebutolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol,indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol,propanolol, sotalol, tertatolol, tilisolol, and timolol, and the like;(3) calcium channel blockers such as amlodipine, aranidipine,azelnidipine, barnidipine, benidipine, bepridil, cinaldipine,clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine,lacidipine, lemildipine, lercanidipine, nicardipine, nifedipine,nilvadipine, nimodepine, nisoldipine, nitrendipine, manidipine,pranidipine, and verapamil, and the like; (4) angiotensin convertingenzyme (ACE) inhibitors such as benazepril; captopril; ceranapril;cilazapril; delapril; enalapril; enalopril; fosinopril; imidapril;lisinopril; losinopril; moexipril; quinapril; quinaprilat; ramipril;perindopril; perindropril; quanipril; spirapril; tenocapril;trandolapril, and zofenopril, and the like; (5) neutral endopeptidaseinhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril,sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonistssuch as tezosentan, A308165, and YM62899, and the like; (7) vasodilatorssuch as hydralazine, clonidine, minoxidil, and nicotinyl alcohol, andthe like; (8) angiotensin II receptor antagonists such as aprosartan,candesartan, eprosartan, irbesartan, losartan, olmesartan, pratosartan,tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270,and the like; (9) α/β adrenergic blockers such as nipradilol, arotinololand amosulalol, and the like; (10) alpha 1 blockers, such as terazosin,urapidil, prazosin, tamsulosin, bunazosin, trimazosin, doxazosin,naftopidil, indoramin, WHP 164, and XENOlO, and the like; (11) alpha 2agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine andguanobenz, and the like; (12) aldosterone inhibitors, and the like; and(13) angiopoietin-2-binding agents such as those disclosed inWO03/030833. Specific anti-hypertensive agents that can be used incombination with polypeptides and agonists described herein include, butare not limited to: diuretics, such as thiazides (e.g., chlorthalidone,cyclothiazide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3,which may be prepared as disclosed in U.S. Pat. No. 2,809,194),dichlorophenamide, hydroflumethiazide, indapamide, polythiazide,bendroflumethazide, methyclothazide, polythiazide, trichlormethazide,chlorthalidone, indapamide, metolazone, quinethazone, althiazide (CAS RN5588-16-9, which may be prepared as disclosed in British Patent No.902,658), benzthiazide (CAS RN 91-33-8, which may be prepared asdisclosed in U.S. Pat. No. 3,108,097), buthiazide (which may be preparedas disclosed in British Patent Nos. 861,367), and hydrochlorothiazide),loop diuretics (e.g. bumetanide, ethacrynic acid, furosemide, andtorasemide), potassium sparing agents (e.g. amiloride, and triamterene(CAS Number 396-01-O)), and aldosterone antagonists (e.g. spironolactone(CAS Number 52-01-7), epirenone, and the like); β-adrenergic blockerssuch as Amiodarone (Cordarone, Pacerone), bunolol hydrochloride (CAS RN31969-05-8, Parke-Davis), acebutolol (±N-[3-Acetyl-4-[2-hydroxy-3-[(1methylethyl)amino]propoxy]phenyl]-butanamide, or(±)-3′-Acetyl-4[2-hydroxy-3-(isopropylamino) propoxy]butyranilide),acebutolol hydrochloride (e.g. Sectral®, Wyeth-Ayerst), alprenololhydrochloride (CAS RN 13707-88-5 see Netherlands Patent Application No.6,605,692), atenolol (e.g. Tenormin®, AstraZeneca), carteololhydrochloride (e.g. Cartrol® Filmtab®, Abbott), Celiprolol hydrochloride(CAS RN 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamololhydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No. 4,059,622),labetalol hydrochloride (e.g. Normodyne®, Schering), esmololhydrochloride (e.g. Brevibloc®, Baxter), levobetaxolol hydrochloride(e.g. Betaxon™ Ophthalmic Suspension, Alcon), levobunolol hydrochloride(e.g. Betagan® Liquifilm® with C CAP® Compliance Cap, Allergan), nadolol(e.g. Nadolol, Mylan), practolol (CAS RN 6673-35-4, see also U.S. Pat.No. 3,408,387), propranolol hydrochloride (CAS RN 318-98-9), sotalolhydrochloride (e.g. Betapace AF™, Berlex), timolol(2-Propanol,1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-,hemihydrate, (S)-, CAS RN 91524-16-2), timolol maleate(S)-I-[(1,1-dimethylethyl)amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-2-propanol(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol(2-Propanol,1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amino]-,(±), CAS RN 66722-44-9), bisoprolol fumarate (such as(±)-1-[4-[[2-(1-Methylethoxy)ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate (2:1) (salt), e.g., Zebeta™, Lederle Consumer),nebivalol (2H-1-Benzopyran-2-methanol,aa′-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS RN 99200-09-6see also U.S. Pat. No. 4,654,362), cicloprolol hydrochloride, such2-Propanol,1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride(2-Propanol,1-[1-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride(CAS RN 13071-11-9), diacetolol hydrochloride (Acetamide,N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy][phenyl]-,monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride(Benzamide,2-hydroxy-541-hydroxy-2-(1-methyl-3-phenylpropyl)amino]ethyl]-,monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride(2-Propanol, 1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 59333-90-3), flestolol sulfate (Benzoic acid,2-fluro-,3-[[2-[aminocarbonyl)amino]-dimethylethyl]amino]-2-hydroxypropylester, (+)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalolhydrochloride (Methanesulfonamide,N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl]-, monohydrochloride CAS RN7701-65-7), metoprolol 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN37350-58-6), metoprolol tartrate (such as 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,Lopressor®, Novartis), pamatolol sulfate (Carbamic acid,[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,methyl ester, (±) sulfate (salt) (2:1), CAS RN 59954-01-7), penbutololsulfate(2-Propanol,1-(2-cyclopentylphenoxy)-3-[1,1-dimethyle-thyl)amino]1,(S)-, sulfate (2:1) (salt), CAS RN 38363-32-5), practolol (Acetamide,N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN6673-35-4;) tiprenolol hydrochloride(Propanol,1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-,hydrochloride, (±), CAS RN 39832-43-4), tolamolol (Benzamide,4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl]amino]ethoxyl]-, CAS RN38103-61-6), bopindolol, indenolol, pindolol, propanolol, tertatolol,and tilisolol, and the like; calcium channel blockers such as besylatesalt of amlodipine (such as3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylatebenzenesulphonate, e.g., Norvasc®, Pfizer), clentiazem maleate(1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-(2S-cis)-,(Z)-2-butenedioate (1:1), see also U.S. Pat. No. 4,567,195), isradipine(3,5-Pyridinedicarboxylic acid,4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethylester,(±)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate,see also U.S. Pat. No. 4,466,972); nimodipine (such as is isopropyl(2-methoxyethyl) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,e.g. Nimotop®, Bayer), felodipine (such as ethyl methyl4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-,e.g. Plendil® Extended-Release, AstraZeneca LP), nilvadipine(3,5-Pyridinedicarboxylic acid,2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl methylethyl)ester, also see U.S. Pat. No. 3,799,934), nifedipine (such as 3,5-pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-,dimethyl ester, e.g., Procardia XL® Extended Release Tablets, Pfizer),diltiazem hydrochloride (such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis., e.g., Tiazac®, Forest), verapamilhydrochloride (such as benzeneacetronitrile,(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Isoptin® SR, Knoll Labs), teludipine hydrochloride(3,5-Pyridinedicarboxylic acid,2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propenyl]phenyl]-1,4-dihydro-6-methyl-,diethyl ester, monohydrochloride) CAS RN 108700-03-4), belfosdil(Phosphonic acid, [2-(2-phenoxy ethyl)-1,3-propane-diyl]bis-, tetrabutylester CAS RN 103486-79-9), fostedil (Phosphonic acid,[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN 75889-62-2),aranidipine, azelnidipine, barnidipine, benidipine, bepridil,cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine,lemildipine, lercanidipine, monatepil maleate (1-Piperazinebutanamide,N-(6, 11-dihydrodibenzo(b,e)thiepin-11-yl)4-(4-fluorophenyl)-, (+)-,(Z)-2-butenedioate (1:1) (±)-N-(6,11-Dihydrodibenzo(b,e)thiep-in-1l-yl)-4-(p-fluorophenyl)-1-piperazinebutyramide maleate (1:1) CAS RN132046-06-1), nicardipine, nisoldipine, nitrendipine, manidipine,pranidipine, and the like; T-channel calcium antagonists such asmibefradil; angiotensin converting enzyme (ACE) inhibitors such asbenazepril, benazepril hydrochloride (such as3[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride, e.g., Lotrel®,Novartis), captopril (such as1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril, Mylan,CAS RN 62571-86-2 and others disclosed in U.S. Pat. No. 4,046,889),ceranapril (and others disclosed in U.S. Pat. No. 4,452,790), cetapril(alacepril, Dainippon disclosed in Eur. Therap. Res. 39:671 (1986);40:543 (1986)), cilazapril (Hoffman-LaRoche) disclosed in J. Cardiovasc.Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride(2H-1,2,4-Benzothiadiazine-7-sulfonamide,3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS RN2259-96-3); disclosed in U.S. Pat. No. 4,385,051), enalapril (and othersdisclosed in U.S. Pat. No. 4,374,829), enalopril, enaloprilat,fosinopril, ((such as L-proline,4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) propoxy](4-phenylbutyl)phosphinyl]acetyl]-, sodium salt, e.g., Monopril, Bristol-Myers Squibband others disclosed in U.S. Pat. No. 4,168,267), fosinopril sodium(L-Proline,4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox), imidapril,indolapril (Schering, disclosed in J. Cardiovasc. Pharmacol. 5:643, 655(1983)), lisinopril (Merck), losinopril, moexipril, moexiprilhydrochloride (3-Isoquinolinecarboxylic acid,2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-12,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, (3S)-CAS RN82586-52-5), quinapril, quinaprilat, ramipril (Hoechsst) disclosed in EP79022 and Curr. Ther. Res. 40:74 (1986), perindopril erbumine (such as2S,3aS,7aS-1-[(S)—N—[(S)-1-Carboxybutyljalanyljhexahydrô-indolinecarboxylicacid, 1-ethyl ester, compound with tert-butylamine (1:1), e.g., Aceon®,Solvay), perindopril (Servier, disclosed in Eur. J. din. Pharmacol.31:519 (1987)), quanipril (disclosed in U.S. Pat. No. 4,344,949),spirapril (Schering, disclosed in Acta. Pharmacol. Toxicol. 59 (Supp.5): 173 (1986)), tenocapril, trandolapril, zofenopril (and othersdisclosed in U.S. Pat. No. 4,316,906), rentiapril (fentiapril, disclosedin Clin. Exp. Pharmacol. Physiol. 10:131 (1983)), pivopril, YS980,teprotide (Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL 36,378(Smith Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai, see CA.102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824 (Ciba-Geigy,3-([1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro-2-ox-o-1-(3S)-benzazepine-1acetic acid HCl, see U.K. Patent No. 2103614), CGS 16,617 (Ciba-Geigy,3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,-5-tetrahydro-2-oxo-1H-1-benzazepine-1-ethanoicacid, see U.S. Pat. No. 4,473,575), Ru 44570 (Hoechst, seeArzneimittelforschung 34:1254 (1985)), R 31-2201 (Hoffman-LaRoche seeFEBS Lett. 165:201 (1984)), CI925 (Pharmacologist 26:243, 266 (1984)),WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)), and those disclosedin US2003006922 (paragraph 28), U.S. Pat. No. 4,337,201, U.S. Pat. No.4,432,971 (phosphonamidates); neutral endopeptidase inhibitors such asomapatrilat (Vanlev®), CGS 30440, cadoxatril and ecadotril, fasidotril(also known as aladotril or alatriopril), sampatrilat, mixanpril, andgemopatrilat, AVE7688, ER4030, and those disclosed in U.S. Pat. No.5,362,727, U.S. Pat. No. 5,366,973, U.S. Pat. No. 5,225,401, U.S. Pat.No. 4,722,810, U.S. Pat. No. 5,223,516, U.S. Pat. No. 4,749,688, U.S.Pat. No. 5,552,397, U.S. Pat. No. 5,504,080, U.S. Pat. No. 5,612,359,U.S. Pat. No. 5,525,723, EP0599444, EP0481522, EP0599444, EP0595610,EP0534363, EP534396, EP534492, EP0629627; endothelin antagonists such astezosentan, A308165, and YM62899, and the like; vasodilators such ashydralazine (apresoline), clonidine (clonidine hydrochloride(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-,monohydrochloride CAS RN 4205-91-8), catapres, minoxidil (loniten),nicotinyl alcohol (roniacol), diltiazem hydrochloride (such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis, e.g., Tiazac®, Forest), isosorbide dinitrate(such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil®Titradose®, Wyeth-Ayerst), sosorbide mononitrate (such as1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic nitrate, e.g.,Ismo®, Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetrioltrinitrate, e.g., Nitrostat® Parke-Davis), verapamil hydrochloride (suchas benzeneacetonitrile, (±)-(alpha)[3-[[2-(3,4 dimethoxypheny1)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Covera HS® Extended-Release, Searle), chromonar(which may be prepared as disclosed in U.S. Pat. No. 3,282,938),clonitate (Annalen 1870 155), droprenilamine (which may be prepared asdisclosed in DE2521113), lidoflazine (which may be prepared as disclosedin U.S. Pat. No. 3,267,104); prenylamine (which may be prepared asdisclosed in U.S. Pat. No. 3,152,173), propatyl nitrate (which may beprepared as disclosed in French Patent No. 1,103,113), mioflazinehydrochloride (1-Piperazineacetamide,3-(aminocarbonyl)4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)-,dihydrochloride CAS RN 83898-67-3), mixidine (Benzeneethanamine,3,4-dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine,2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphenethyl)imino]pyrrolidine CAS RN 27737-38-8), molsidomine(1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, innersalt CAS RN 25717-80-0), isosorbide mononitrate (D-Glucitol,1,4:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7), erythrityltetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS RN7297-25-8), clonitrate(1,2-Propanediol, 3-chloro-, dinitrate (7CI, 8CI,9CI) CAS RN 2612-33-1), dipyridamole Ethanol,2,2′,2″,2″-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]tetrakis-CASRN 58-32-2), nicorandil (CAS RN 65141-46-0 3-), pyridinecarboxamide(N-[2-(nitrooxy)ethyl]-Nisoldipine3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl esterCAS RN 63675-72-9), nifedipine3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN21829-25-4), perhexiline maleate (Piperidine,2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN 6724-53-4),oxprenolol hydrochloride (2-Propanol,1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-, hydrochlorideCAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),verapamil (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-CAS RN 52-53-9) and the like; angiotensinII receptor antagonists such as, aprosartan, zolasartan, olmesartan,pratosartan, FI6828K, RNH6270, candesartan(1H-Benzimidazole-7-carboxylic acid,2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]4-yl]methyl]-CAS RN139481-59-7), candesartan cilexetil((+/−)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]-1H-benzimidazolecarboxylate, CAS RN 145040-37-5, U.S. Pat. No. 5,703,110 and U.S. Pat.No. 5,196,444), eprosartan(3-[1-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)propenoic acid, U.S. Pat. No. 5,185,351 and U.S. Pat. No. 5,650,650),irbesartan(2-n-butyl-3-[[2′-(lh-tetrazol-5-yl)biphenyl-4-yl]methyl]1,3-diazazspiro[4,4]non-1-en-4-one,U.S. Pat. No. 5,270,317 and U.S. Pat. No. 5,352,788), losartan(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole,potassium salt, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 andU.S. Pat. No. 5,128,355), tasosartan(5,8-dihydro-2,4-dimethyl-8-[(2′-(1H-tetrazol-5-yl)[1,r-biphenyl]4-yl)methyl]-pyrido[2,3-d]pyrimidin-7(6H)-one,U.S. Pat. No. 5,149,699), telmisartan(4′-[(1,4-dimethyl-2′-propyl-(2,6′-bi-1H-benzimidazol)-r-yl)]-[1,1′-biphenyl]-2-carboxylic acid, CAS RN 144701-48-4, U.S. Pat. No.5,591,762), milfasartan, abitesartan, valsartan (Diovan® (Novartis),(S)—N-valeryl-N-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine, U.S.Pat. No. 5,399,578), EXP-3137(2-N-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylicacid, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 and U.S. Pat. No.5,128,355),3-(2′-(tetrazol-5-yl)-1,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine,4′[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-1,r-biphenyl]-2-carboxylicacid,2-butyl-6-(1-methoxy-1-methylethyl)-2-[2′-)1H-tetrazol-5-yl)biphenyl-4-ylmethyl]guinazolin-4(3H)-one,3-[2′-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5-b]pyridine,2-butyl-4-chloro-1-[(2′-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carboxylicacid, 2-butyl-4-chloro-1-[[2′-(1H-tetrazol-5-yl) [1,1′-biphenyl]-4-yl]methyl]-1H-imidazole-5-carboxylicacid-1-(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,1′-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol-5-ylphenyl)]pyridine,6-butyl-2-(2-phenylethyl)-5[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-methyl]pyrimidin-4-(3H)-one D,Llysine salt,5-methyl-7-n-propyl-8-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one,2,7-diethyl-5-[[2′-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]triazolepotassium salt,2-[2-butyl-4,5-dihydro-4-oxo-3-[2′-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoicacid, ethyl ester, potassium salt,3-methoxy-2,6-dimethyl-4-[[2′(1H-tetrazol-5-yl)-1,1′-biphenyl-4-yl]methoxy]pyridine,2-ethoxy-1-[[2′-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylicacid,1-[N-(2′-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)cyclopentane-1-carboxylicacid, 7-methyl-2n-propyl-3-[[2′1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine,2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quinolinyl]sodiumbenzoate, 2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2′-(IH-tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine,2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]benzoicacid tetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol-1-yl]octanoyl]-L-proline,1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one,5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2′(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9-one,44142′-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetrahydro-2-trifylquinazoline,2-(2-chlorobenzoyl)imino-5-ethyl-3-[2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl-1,3,4-thiadiazoline,2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline-2-ylidene]aminocarbonyl-1-cyclopentencarboxylicacid dipotassium salt, and2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidzole-5-carboxylicacid 1-ethoxycarbonyloxyethyl ester, those disclosed in patentpublications EP475206, EP497150, EP539086, EP539713, EP535463, EP535465,EP542059, EP497121, EP535420, EP407342, EP415886, EP424317, EP435827,EP433983, EP475898, EP490820, EP528762, EP324377, EP323841, EP420237,EP500297, EP426021, EP480204, EP429257, EP430709, EP434249, EP446062,EP505954, EP524217, EP514197, EP514198, EP514193, EP514192, EP450566,EP468372, EP485929, EP503162, EP533058, EP467207 EP399731, EP399732,EP412848, EP453210, EP456442, EP470794, EP470795, EP495626, EP495627,EP499414, EP499416, EP499415, EP511791, EP516392, EP520723, EP520724,EP539066, EP438869, EP505893, EP530702, EP400835, EP400974, EP401030,EP407102, EP411766, EP409332, EP412594, EP419048, EP480659, EP481614,EP490587, EP467715, EP479479, EP502725, EP503838, EP505098, EP505111EP513,979 EP507594, EP510812, EP511767, EP512675, EP512676, EP512870,EP517357, EP537937, EP534706, EP527534, EP540356, EP461040, EP540039,EP465368, EP498723, EP498722, EP498721, EP515265, EP503785, EP501892,EP519831, EP532410, EP498361, EP432737, EP504888, EP508393, EP508445,EP403159, EP403158, EP425211, EP427463, EP437103, EP481448, EP488532,EP501269, EP500409, EP540400, EP005528, EP028834, EP028833, EP411507,EP425921, EP430300, EP434038, EP442473, EP443568, EP445811, EP459136,EP483683, EP518033, EP520423, EP531876, EP531874, EP392317, EP468470,EP470543, EP502314, EP529253, EP543263, EP540209, EP449699, EP465323,EP521768, EP415594, WO92/14468, WO93/08171, WO93/08169, WO91/00277,WO91/00281, WO91/14367, WO92/00067, WO92/00977, WO92/20342, WO93/04045,WO93/04046, WO91/15206, WO92/14714, WO92/09600, WO92/16552, WO93/05025,WO93/03018, WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909,WO91/12001, WO91/11999, WO91/15209, WO91/15479, WO92/20687, WO92/20662,WO92/20661, WO93/01177, WO91/14679, WO91/13063, WO92/13564, WO91/17148,WO91/18888, WO91/19715, WO92/02257, WO92/04335, WO92/05161, WO92/07852,WO92/15577, WO93/03033, WO91/16313, WO92/00068, WO92/02510, WO92/09278,WO9210179, WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183,WO92/10182, WO92/10187, WO92/10184, WO92/10188, WO92/10180, WO92/10185,WO92/20651, WO93/03722, WO93/06828, WO93/03040, WO92/19211, WO92/22533,WO92/06081, WO92/05784, WO93/00341, WO92/04343, WO92/04059, U.S. Pat.No. 5,104,877, U.S. Pat. No. 5,187,168, U.S. Pat. No. 5,149,699, U.S.Pat. No. 5,185,340, U.S. Pat. No. 4,880,804, U.S. Pat. No. 5,138,069,U.S. Pat. No. 4,916,129, U.S. Pat. No. 5,153,197, U.S. Pat. No.5,173,494, U.S. Pat. No. 5,137,906, U.S. Pat. No. 5,155,126, U.S. Pat.No. 5,140,037, U.S. Pat. No. 5,137,902, U.S. Pat. No. 5,157,026, U.S.Pat. No. 5,053,329, U.S. Pat. No. 5,132,216, U.S. Pat. No. 5,057,522,U.S. Pat. No. 5,066,586, U.S. Pat. No. 5,089,626, U.S. Pat. No.5,049,565, U.S. Pat. No. 5,087,702, U.S. Pat. No. 5,124,335, U.S. Pat.No. 5,102,880, U.S. Pat. No. 5,128,327, U.S. Pat. No. 5,151,435, U.S.Pat. No. 5,202,322, U.S. Pat. No. 5,187,159, U.S. Pat. No. 5,198,438,U.S. Pat. No. 5,182,288, U.S. Pat. No. 5,036,048, U.S. Pat. No.5,140,036, U.S. Pat. No. 5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat.No. 5,153,347, U.S. Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, U.S.Pat. No. 5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. No. 5,208,234,U.S. Pat. No. 5,208,235, U.S. Pat. No. 5,212,195, U.S. Pat. No.5,130,439, U.S. Pat. No. 5,045,540, U.S. Pat. No. 5,041,152, and U.S.Pat. No. 5,210,204, and pharmaceutically acceptable salts and estersthereof; α/β adrenergic blockers such as nipradilol, arotinolol,amosulalol, bretylium tosylate (CAS RN: 61-75-6), dihydroergtaminemesylate (such as ergotaman-3′,6′,18-trione,9,-10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-,(5′(α))-,monomethanesulfonate, e.g., DHE 45® Injection, Novartis), carvedilol(such as(±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol,e.g., Coreg®, SmithKline Beecham), labetalol (such as5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino]ethyl]salicylamidemonohydrochloride, e.g., Normodyne®, Schering), bretylium tosylate(Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt with4-methylbenzenesulfonic acid (1:1) CAS RN 61-75-6), phentolaminemesylate (Phenol,3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-,monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate(5H-1,3-Dioxolo[4,5-f]indole,74244-(2-methoxyphenyl)-1-piperazinyflethylb(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5), zolertinehydrochloride (Piperazine, 1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-,monohydrochloride (8Cl, 9Cl) CAS RN 7241-94-3) and the like; aadrenergic receptor blockers, such as alfuzosin (CAS RN: 81403-68-1),terazosin, urapidil, prazosin (Minipress®), tamsulosin, bunazosin,trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOlO,fenspiride hydrochloride (which may be prepared as disclosed in U.S.Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and labetalolhydrochloride and combinations thereof; a 2 agonists such as methyldopa,methyldopa HCL, lofexidine, tiamenidine, moxonidine, rilmenidine,guanobenz, and the like; aldosterone inhibitors, and the like; renininhibitors including Aliskiren (SPP100; Novartis/Speedel);angiopoietin-2-binding agents such as those disclosed in WO03/030833;anti-angina agents such as ranolazine (hydrochloride1-Piperazineacetamide,N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride(2-Propanol,1-[4-[2(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride (Methanone,[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,monohydrochloride CAS RN 62134-34-3), cinepazetmaleatel-Piperazineacetic acid,4-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7), tosifen(Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN32295-184), verapamilhydrochloride (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-,monohydrochloride CAS RN 152-114), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), and ranolazine hydrochloride (1-Piperazineacetamide,N-(2,6-dimethylphenyl)4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]-CAS RN32295-184); adrenergic stimulants such as guanfacine hydrochloride (suchas N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride, e.g.,Tenex® Tablets available from Robins); methyldopa-hydrochlorothiazide(such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined withHydrochlorothiazide (such as6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide1,1-dioxide, e.g., the combination as, e.g., Aldoril® Tablets availablefrom Merck), methyldopa-chlorothiazide (such as 6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and methyldopa asdescribed above, e.g., Aldoclor®, Merck), clonidine hydrochloride (suchas 2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride andchlorthalidone (such as 2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl)benzenesulfonamide), e.g., Combipres®, Boehringer Ingelheim), clonidinehydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazolinehydrochloride, e.g., Catapres®, Boehringer Ingelheim), clonidine(1H-Imidazol-2-amine, N-(2,6-dichlorophenyl)4,5-dihydro-CAS RN4205-90-7), Hyzaar (Merck; a combination of losartan andhydrochlorothiazide), Co-Diovan (Novartis; a combination of valsartanand hydrochlorothiazide, Lotrel (Novartis; a combination of benazepriland amlodipine) and Caduet (Pfizer; a combination of amlodipine andatorvastatin), and those agents disclosed in US20030069221.

1.3.2.11 Agents for the Treatment of Respiratory Disorders

The GCC agonist peptides described herein can be used in combinationtherapy with one or more of the following agents useful in the treatmentof respiratory and other disorders including but not limited to: (1)β-agonists including but not limited to: albuterol (PRO VENTIL®, S ALBUTAMO1®, VENTOLIN®), bambuterol, bitoterol, clenbuterol, fenoterol,formoterol, isoetharine (BRONKOSOL®, BRONKOMETER®), metaproterenol(ALUPENT®, METAPREL®), pirbuterol (MAXAIR®), reproterol, rimiterol,salmeterol, terbutaline (BRETHAIRE®, BRETHINE®, BRICANYL®), adrenalin,isoproterenol (ISUPREL®), epinephrine bitartrate (PRIMATENE®),ephedrine, orciprenline, fenoterol and isoetharine; (2) steroids,including but not limited to beclomethasone, beclomethasonedipropionate, betamethasone, budesonide, bunedoside, butixocort,dexamethasone, flunisolide, fluocortin, fluticasone, hydrocortisone,methyl prednisone, mometasone, predonisolone, predonisone, tipredane,tixocortal, triamcinolone, and triamcinolone acetonide; (3)β2-agonist-corticosteroid combinations [e.g., salmeterol-fluticasone (ADV AIR®), formoterol-budesonid (S YMBICORT®)]; (4) leukotriene D4receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., anycompound that is capable of blocking, inhibiting, reducing or otherwiseinterrupting the interaction between leukotrienes and the Cys LTIreceptor) including but not limited to: zafhiukast, montelukast,montelukast sodium (SINGULAIR®), pranlukast, iralukast, pobilukast,SKB-106,203 and compounds described as having LTD4 antagonizing activitydescribed in U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase inhibitorsand/or leukotriene biosynthesis inhibitors [e.g., zileuton and BAY1005(CA registry 128253-31-6)]; (6) histamine Ell receptorantagonists/antihistamines (i.e., any compound that is capable ofblocking, inhibiting, reducing or otherwise interrupting the interactionbetween histamine and its receptor) including but not limited to:astemizole, acrivastine, antazoline, azatadine, azelastine, astamizole,bromopheniramine, bromopheniramine maleate, carbinoxamine, carebastine,cetirizine, chlorpheniramine, chloropheniramine maleate, cimetidineclemastine, cyclizine, cyproheptadine, descarboethoxyloratadine,dexchlorpheniramine, dimethindene, diphenhydramine, diphenylpyraline,doxylamine succinate, doxylarnine, ebastine, efletirizine, epinastine,famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen,levocabastine, levocetirizine, levocetirizine, loratadine, meclizine,mepyramine, mequitazine, methdilazine, mianserin, mizolastine,noberastine, norasternizole, noraztemizole, phenindamine, pheniramine,picumast, promethazine, pynlamine, pyrilamine, ranitidine, temelastine,terfenadine, trimeprazine, tripelenamine, and triprolidine; (7) ananticholinergic including but not limited to: atropine, benztropine,biperiden, flutropium, hyoscyamine (e.g. Levsin®; Levbid®; Levsin/SL®,Anaspaz®, Levsinex Timecaps®, NuLev®), ilutropium, ipratropium,ipratropium bromide, methscopolamine, oxybutinin, rispenzepine,scopolamine, and tiotropium; (8) an anti-tussive including but notlimited to: dextromethorphan, codeine, and hydromorphone; (9) adecongestant including but not limited to: pseudoephedrine andphenylpropanolamine; (10) an expectorant including but not limited to:guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerolguaicolate, and iodinated glycerol; (11) a bronchodilator including butnot limited to: theophylline and aminophylline; (12) ananti-inflammatory including but not limited to: fluribiprofen,diclophenac, indomethacin, ketoprofen, S-ketroprophen, tenoxicam; (13) aPDE (phosphodiesterase) inhibitor including but not limited to thosedisclosed herein; (14) a recombinant humanized monoclonal antibody [e.g.xolair (also called omalizumab), rhuMab, and talizumab]; (15) ahumanized lung surfactant including recombinant forms of surfactantproteins SP-B, SP-C or SP-D [e.g. SURFAXIN®, formerly known as dsc-104(Discovery Laboratories)], (16) agents that inhibit epithelial sodiumchannels (ENaC) such as amiloride and related compounds; (17)antimicrobial agents used to treat pulmonary infections such asacyclovir, amikacin, amoxicillin, doxycycline, trimethoprinsulfamethoxazole, amphotericin B, azithromycin, clarithromycin,roxithromycin, clarithromycin, cephalosporins(ceffoxitin, cefmetazoleetc), ciprofloxacin, ethambutol, gentimycin, ganciclovir, imipenem,isoniazid, itraconazole, penicillin, ribavirin, rifampin, rifabutin,amantadine, rimantidine, streptomycin, tobramycin, and vancomycin; (18)agents that activate chloride secretion through Ca++ dependent chloridechannels (such as purinergic receptor (P2Y(2) agonists); (19) agentsthat decrease sputum viscosity, such as human recombinant DNase 1,(Pulmozyme®); (20) nonsteroidal anti-inflammatory agents (acemetacin,acetaminophen, acetyl salicylic acid, alclofenac, alminoprofen, apazone,aspirin, benoxaprofen, bezpiperylon, bucloxic acid, carprofen, clidanac,diclofenac, diclofenac, diflunisal, diflusinal, etodolac, fenbufen,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, flufenisal, fluprofen, flurbiprofen,flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketoprofen,ketorolac, meclofenamic acid, meclofenamic acid, mefenamic acid,mefenamic acid, miroprofen, mofebutazone, nabumetone oxaprozin,naproxen, naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone,phenacetin, phenylbutazone, phenylbutazone, piroxicam, piroxicam,pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine, sulindac,sulindac, suprofen, tiaprofenic acid, tiopinac, tioxaprofen, tolfenamicacid, tolmetin, tolmetin, zidometacin, zomepirac, and zomepirac); and(21) aerosolized antioxidant therapeutics such as S-Nitrosoglutathione.

1.3.2.12 Anti-Diabetic Agents

The GCC agonist peptides described herein can be used in therapeuticcombination with one or more anti-diabetic agents, including but notlimited to: PPARy agonists such as glitazones (e.g., WAY-120,744, AD5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer),englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555(Mitsibishi disclosed in U.S. Pat. No. 5,594,016), pioglitazone (such assuch as Actos™ pioglitazone; Takeda), rosiglitazone (Avandia™; SmithKline Beecham), rosiglitazone maleate, troglitazone (Rezulin®, disclosedin U.S. Pat. No. 4,572,912), rivoglitazone (CS-01 1, Sankyo), GL-262570(Glaxo Welcome), BRL49653 (disclosed in WO98/05331), CLX-0921, 5-BTZD,GW-0207, LG-100641, JJT-501 (JPNT/P&U), L-895645 (Merck), R-119702(Sankyo/Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512,LY-519818, R483 (Roche), T131 (Tularik), and the like and compoundsdisclosed in U.S. Pat. No. 4,687,777, U.S. Pat. No. 5,002,953, U.S. Pat.No. 5,741,803, U.S. Pat. No. 5,965,584, U.S. Pat. No. 6,150,383, U.S.Pat. No. 6,150,384, U.S. Pat. No. 6,166,042, U.S. Pat. No. 6,166,043,U.S. Pat. No. 6,172,090, U.S. Pat. No. 6,211,205, U.S. Pat. No.6,271,243, U.S. Pat. No. 6,288,095, U.S. Pat. No. 6,303,640, U.S. Pat.No. 6,329,404, U.S. Pat. No. 5,994,554, WO97/10813, WO97/27857,WO97/28115, WO97/28137, WO97/27847, WO00/76488, WO03/000685,WO03/027112, WO03/035602, WO03/048130, WO03/055867, and pharmaceuticallyacceptable salts thereof; biguanides such as metformin hydrochloride(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such asGlucophage™, Bristol-Myers Squibb); metformin hydrochloride withglyburide, such as Glucovance™, Bristol-Myers Squibb); buformin(Imidodicarbonimidic diamide, N-butyl-); etoformine(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms(including where the salt is chosen from the group of, acetate,benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate,palmoate, aspartate, methanesulphonate, maleate,parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate,tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate,hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate,paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutamate,pyrrolidonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,nitrate, sulphite, dithionate and phosphate), and phenformin; proteintyrosine phosphatase-IB (PTP-IB) inhibitors, such as A-401,674, KR61639, OC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715,and those disclosed in WO99/585521, WO99/58518, WO99/58522, WO99/61435,WO03/032916, WO03/032982, WO03/041729, WO03/055883, WO02/26707,WO02/26743, JP2002114768, and pharmaceutically acceptable salts andesters thereof; sulfonylureas such as acetohexamide (e.g. Dymelor, EliLilly), carbutamide, chlorpropamide (e.g. Diabinese®, Pfizer),gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada Inc),glimepiride (e.g. disclosed in U.S. Pat. No. 4,379,785, such as Amaryl,Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL ExtendedRelease, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide (e.g.Micronase or Glynase Prestab, Pharmacia & Upjohn and Diabeta, Aventis),tolazamide (e.g. Tolinase), and tolbutamide (e.g. Orinase), andpharmaceutically acceptable salts and esters thereof; meglitinides suchas repaglinide (e.g. Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), andnateglinide (e.g. Starlix®, Novartis), and pharmaceutically acceptablesalts and esters thereof; a glucoside hydrolase inhibitors (or glucosideinhibitors) such as acarbose (e.g. Precose™, Bayer disclosed in U.S.Pat. No. 4,904,769), miglitol (such as GLYSET™, Pharmacia & Upjohndisclosed in U.S. Pat. No. 4,639,436), camiglibose (Methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 14,and the compounds disclosed in U.S. Pat. No. 4,062,950, U.S. Pat. No.4,174,439, U.S. Pat. No. 4,254,256, U.S. Pat. No. 4,701,559, U.S. Pat.No. 4,639,436, U.S. Pat. No. 5,192,772, U.S. Pat. No. 4,634,765, U.S.Pat. No. 5,157,116, U.S. Pat. No. 5,504,078, U.S. Pat. No. 5,091,418,U.S. Pat. No. 5,217,877, US51091 and WOO 1/47528 (polyamines); α-amylaseinhibitors such as tendamistat, trestatin, and Al-3688, and thecompounds disclosed in U.S. Pat. No. 4,451,455, U.S. Pat. No. 4,623,714,and U.S. Pat. No. 4,273,765; SGLT2 inhibtors including those disclosedin U.S. Pat. No. 6,414,126 and U.S. Pat. No. 6,515,117; an aP2 inhibitorsuch as disclosed in U.S. Pat. No. 6,548,529; insulin secreatagoguessuch as linogliride, A-4166, forskilin, dibutyrl cAMP,isobutylmethylxanthine (IBMX), and pharmaceutically acceptable salts andesters thereof; fatty acid oxidation inhibitors, such as clomoxir, andetomoxir, and pharmaceutically acceptable salts and esters thereof; A2antagonists, such as midaglizole, isaglidole, deriglidole, idazoxan,earoxan, and fluparoxan, and pharmaceutically acceptable salts andesters thereof; insulin and related compounds (e.g. insulin mimetics)such as biota, LP-100, novarapid, insulin detemir, insulin lispro,insulin glargine, insulin zinc suspension (lente and ultralente),Lys-Pro insulin, GLP-I (1-36) amide, GLP-I (73-7) (insulintropin,disclosed in U.S. Pat. No. 5,614,492), LY-315902 (Lilly), GLP-I(7-36)—NH2), AL-401 (Autoimmune), certain compositions as disclosed inU.S. Pat. No. 4,579,730, U.S. Pat. No. 4,849,405, U.S. Pat. No.4,963,526, U.S. Pat. No. 5,642,868, U.S. Pat. No. 5,763,396, U.S. Pat.No. 5,824,638, U.S. Pat. No. 5,843,866, U.S. Pat. No. 6,153,632, U.S.Pat. No. 6,191,105, and WO 85/05029, and primate, rodent, or rabbitinsulin including biologically active variants thereof including allelicvariants, more preferably human insulin available in recombinant form(sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin), alsosee the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins); non-thiazolidinediones such as JT-501 and farglitazar(GW-2570/GI-262579), and pharmaceutically acceptable salts and estersthereof; PPARα/γ dual agonists such as AR-H039242 (Aztrazeneca),GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536, GW-1929,GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxothiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl]methyljbenzamide),L-796449, LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar(BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501) and thosedisclosed in WO99/16758, WO99/19313, WO99/20614, WO99/38850, WO00/23415,WO00/23417, WO00/23445, WO00/50414, WO01/00579, WO01/79150, WO02/062799,WO03/004458, WO03/016265, WO03/018010, WO03/033481, WO03/033450,WO03/033453, WO03/043985, WO 031053976, U.S. application Ser. No.09/664,598, filed Sep. 18, 2000, Murakami et al. Diabetes 47, 1841-1847(1998), and pharmaceutically acceptable salts and esters thereof; otherinsulin sensitizing drugs; VPAC2 receptor agonists; GLK modulators, suchas those disclosed in WO03/015774; retinoid modulators such as thosedisclosed in WO03/000249; GSK 3β/GSK 3 inhibitors such as4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine andthose compounds disclosed in WO03/024447, WO03/037869, WO03/037877,WO03/037891, WO03/068773, EP1295884, EP1295885, and the like; glycogenphosphorylase (HGLPa) inhibitors such as CP-368,296, CP-316,819,BAYR3401, and compounds disclosed in WOO 1/94300, WO02/20530,WO03/037864, and pharmaceutically acceptable salts or esters thereof;ATP consumption promotors such as those disclosed in WO03/007990; TRB3inhibitors; vanilloid receptor ligands such as those disclosed inWO03/049702; hypoglycemic agents such as those disclosed in WO03/015781and WO03/040114; glycogen synthase kinase 3 inhibitors such as thosedisclosed in WO03/035663 agents such as those disclosed in WO99/51225,US20030134890, WO01/24786, and WO03/059870; insulin-responsive DNAbinding protein-1 (IRDBP-I) as disclosed in WO03/057827, and the like;adenosine A2 antagonists such as those disclosed in WO03/035639,WO03/035640, and the like; PPARδ agonists such as GW 501516, GW 590735,and compounds disclosed in JP10237049 and WO02/14291; dipeptidylpeptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide,NVP-DPP728A(1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine,disclosed by Hughes et al, Biochemistry, 38(36), 11597-11603, 1999),P32/98, NVP-LAF-237, P3298, TSL225(tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, disclosedby Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540), valinepyrrolidide, TMC-2A/2B/2C, CD-26 inhibitors, FE999011, P9310/K364, VIP0177, DPP4, SDZ 274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides asdisclosed by Ashworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22,pp 1163-1166 and 2745-2748 (1996), and the compounds disclosed in U.S.Pat. No. 6,395,767, U.S. Pat. No. 6,573,287, U.S. Pat. No. 6,395,767(compounds disclosed include BMS-477118, BMS-471211 and BMS 538,305),WO99/38501, WO99/46272, WO99/67279, WO99/67278, WO99/61431WO03/004498,WO03/004496, EP1258476, WO02/083128, WO02/062764, WO03/000250,WO03/002530, WO03/002531, WO03/002553, WO03/002593, WO03/000180, andWO03/000181; GLP-I agonists such as exendin-3 and exendin-4 (includingthe 39 aa polypeptide synthetic exendin-4 called Exenatide®), andcompounds disclosed in US2003087821 and NZ 504256, and pharmaceuticallyacceptable salts and esters thereof; peptides including amlintide andSymlin® (pramlintide acetate); and glycokinase activators such as thosedisclosed in US2002103199 (fused heteroaromatic compounds) andWO02/48106 (isoindolin-1-one-substituted propionamide compounds).

Examples Example 1: Clinical Study for Safety and Efficacy in Humans forthe Treatment of Chronic Idiopathic Constipation

A randomized, double-blind, placebo-controlled, 14-day repeat oral, doseranging study was conducted in patients with chronic idiopathicconstipation (CIC). The primary objective of this study was to evaluatethe safety of SP-304 (1.0 mg, 3.0 mg, 9.0 mg and 0.3 mg) for 14 days inpatients with CIC. One secondary objective was to assess thepharmacokinetic profile of SP-304 in plasma. Other secondary objectivesincluded evaluations of pharmacodynamic effects (efficacy) on parameterssuch as the time to first bowel movement after daily dosing with SP-304,bowel habits over time—for example, spontaneous bowel movements (SBMs),complete spontaneous bowel movements (CSBMs), and stool consistency[using Bristol Stool Form Scale (BSFS)]—and other patient reportedoutcomes such as abdominal discomfort.

The study included five arms with assigned interventions as indicated inthe table below.

Arms Interventions SP-304 1.0 mg: Experimental Subjects receiving SP-3041.0 mg for 14 consecutive days SP-304 3.0 mg: Experimental Subjectsreceiving SP-304 3.0 mg for 14 consecutive days SP-304 9.0 mg:Experimental Subjects receiving SP-304 9.0 mg for 14 consecutive daysPlacebo: Placebo Comparator Subjects receiving Placebo for 14consecutive days SP-304 0.3 mg: Experimental Subjects receiving SP-3040.3 mg for 14 consecutive days

Subjects diagnosed with CIC were screened for the anticipated 4 cohortsto yield 80 randomized subjects for enrollment. There were four dosecohorts (1.0 mg, 3.0 mg, 9.0 mg and 0.3 mg) with 20 subjects per dosecohort [randomization ratio 3:1 (15 receive SP-304:5 receive placebo)].Subjects who continued to meet all the entry criteria and complete thepre-treatment bowel movement (BM) diary received, in a double-blind,randomized fashion, SP-304 or matching placebo. The entry criteriaincluded (1) meeting modified ROME III criteria for chronic constipation(CC); (2) no significant finding in colonoscopy within past 5 years; (3)good health as determined by physical examination, medical history,vital signs, ECG, clinical chemistry, hematology, urinalysis, drugscreen and serology assessments; and (4) during 14-day pre-treatmentperiod, subjects reporting <6 SBM and <3 CSBM in each pre-treatmentweek. All subjects receiving at least one dose of SP-304 or matchingplacebo were considered evaluable for the safety endpoints (78 total).If a subject did not have a major protocol deviation, had at least 5days of study treatment each week and corresponding entries for bowelhabits, he/she was considered evaluable for efficacy parameters (54-55total).

The demographics of the subjects in the study are summarized in thetable below.

Placebo 0.3 mg 1.0 mg 3.0 mg 9.0 mg Age 47.7 (14.6) 51.1 (12.0) 50.5(10.6) 48.5 (16.1) 47.3 (12.7) Gender Female   18 (90.0%)   12 (85.7%)  14 (100%)   13 (86.7)   12 (80%) Male   2 (10.0%)   2 (14.3%)   0   2(13.3%)   3 (20%) Race White   17 (85.0%)   13 (92.9%)   12 (85.7%)   14(93.3%)   12 (80.0%) African American   1 (5.0%)   0   1 (7.1%)   0   2(13.3%) Asian   1 (5.0%)   1 (7.1%)   1 (7.1%)   0   1 (6.7%) AmericanIndian   1 (5.0%)   0   0   0   0 Other   0   0   0   1 (6.7%)   0Values for age are the mean (standard deviation); values for gender andrace are the number (percentage of experimental arm).

Results

Pharmacokinetics and Safety:

There was no detectable systemic absorption of plecanatide (assaysensitivity ≧10 ng/mL). No serious adverse events (SAE) were reported insubjects receiving plecanatide and no deaths reported in this study. 10%(2/20) subjects who received placebo and 17.2% (10/58) subjects whoreceived SP-304 reported adverse events considered as related to thetreatment. The majority of adverse events were mild/moderate andtransient in nature. 10% (2/20) subjects who received placebo and 5.2%(3/58) subjects who received SP-304 reported GI-related adverse eventsconsidered as related to treatment. There was no diarrhea reported forany subject receiving SP-304. The table below is a GI-related adverseevent (AE) summary.

Placebo 0.3 mg 1.0 mg 3.0 mg 9.0 mg n = 20 n = 14 n = 14 n = 15 n = 15Abdominal 1 (5.0%) 0 0 0 0 Cramping Abdominal 1 (5.0%) 0 0 0 0 PainBloating 0 0 0 0 1 (6.7%) Diarrhea 1 (5.0%) 0 0 0 0 Flatulence  2(10.0%) 0 0 0 0 Nausea 0 1 (7.1%) 0 0 Upset 0 0 0 1 (6.7%) 0 StomachValues are the number (percentage of experimental arm).

Efficacy:

SP-304 (plecanatide) treatment decreased the time to first bowelmovement, increased stool frequency (SBM and CSBM), improved stoolconsistency, and reduced straining and abdominal discomfort. See FIGS.1-6.

Example 2: Composition of Wet Granulation Batch 10005

Item No. Ingredient Use Concentration % w/w 1 SP304 0.23 2 Mannogem EZ,Diluent 79.77 USP/EP (Mannitol) 3 PROSOLV SMCC 90 Binder 15.0 LM(silicified microcrystalline cellulose) 4 Purified Water vehicle n/a(chilled to 5° C.), USP 5 Purified Water n/a (chilled to 5° C.), USP 6Explotab (Sodium Disintregant 4.0 Starch Glycolate) 7 Pruv (sodiumstearyl Lubricant 1.0 fumarate) Total 100

Example 3: Composition of Wet Granulation Batch 10007

Item No. Ingredient Use Concentration % w/w 1 SP304 0.3 3 PROSOLV SMCC90 Binder 95.7 HD (silicified microcrystalline cellulose) 4 PurifiedWater vehicle n/a (chilled to 5° C.), USP 5 Purified Water n/a (chilledto 5° C.), USP 6 Explotab (Sodium Disintregant 4.0 Starch Glycolate)Total 100

Example 4: Excipient Compatibility

Binary mixtures of SP-304 were prepared and stored in glass vials. Forsolid excipients the binary mixtures were comprised of 9.1% or 50%excipient. Glass vials were stored at 40 C/75RH open or closed. Thepercent purity (measured by HPLC) of the GCC agonist peptide (SP-304)after storage for the time indicated in each column (i.e., 1, 2, or 3months for the closed vial and 0.5, 1, 2, or 3 months for the openvials) is indicated by numerical values.

Closed Open PURPOSE EXCIPIENT 1 M 2 M 3 M 0.5 M 1 M 2 M 3 M None None91.4 88.2 84.1 93.7 91.2 88.2 84.8 Diluent Sorbitol 92.4 90.1 87.2 92.290.8 87.1 80.9 Mannitol 91.9 88.4 85.1 92.6 90.5 87.9 83.8 Prosolv 92.289.6 86.3 93 90.5 87.8 83.7 Starch 91.4 88.7 85.4 92.5 90.5 87.9 83.7Binder Emdex 91.3 88.7 85.2 91.8 90.7 87.9 81.9 Plasdone 92.8 90.6 85.693.1 90.4 87.3 83 Disintegrant Explotab 91.9 89.4 87.1 92.2 90.3 84.778.3 Polyplasdone 92 89 85.6 93.5 90.3 87.4 83.1 Glidant Cabosil 92.188.3 85.6 92.6 90.5 87.3 84 Lubricant Mg stearte 91.5 87.7 84.6 92.690.6 87.6 83.8 PRUV 92 88.3 85.7 92.2 90.5 87.5 83.8 compritol 90.8 87.184.4 92 90.5 86.7 84.1 Excipient PEG 3350 90.9 87 83.3 91.5 89.4 84.477.5 Antioxidant Ascorbic acid 91.3 86.9 83 92.8 90 85.7 83.8 BHA 91.988.9 85.9 93.5 90.8 87.4 85.8 BHT 90.8 87.2 84.6 92.4 90.3 86.6 83.6EDTA 90.9 87.5 84.1 92.3 90.4 86.7 84.6 Capsule HPMC capsule 92.2 8985.2 92.3 90.2 86.4 83.5 Gelatin capsule 91.5 88.3 84.3 84.3 90.5 86.783.6 Liquid for liquid filled Medium chain trig 90.4 capsule PGdicaprylocaprate 89.3 Vit E 90 Soybean oil 89.6 Cremaphor 79.7 PG 3.4 PG400 0.7

Example 5: Geometric Dry Mix for 0.3 mg Capsule

Place 12 g mannitol in mortar. Add 4 g SP-304 and gently mix until avisually uniform powder is obtained. Transfer to Turbula mixer. Rinsemortar with mannitol and transfer to Turbula mixer and mix at high speedfor 10 minutes. Add about 150 g of mannitol to 4 quart V-shell mixer.Transfer the contents of the Turbula mixer to the V-shell and add 150 gof mannitol mix. Discharge v-shell contents and screen through 40 meshand return to mixer. Add 586 g of mannitol to mixer and mix for 20minutes.

Example 6: Wet Granulation Process

Batch 017-10005 comprised of mannitol and low-moisture (2.4%) PROSOLVLM90 (0.33 g/mL) was sprayed with SP-304 solution and fluid bed driedresulted in granulation water content of 0.35%. The final blendcontained 1% water, flowed well, and filled capsules well. The 2ndprototype 017-1006 comprised of the same components was adjusted toobtain a target capsule fill weight of 100 mg based on the results ofthe 1st batch. Water was sprayed onto powder blend with SP-304. Theinlet temperature was 50 C and the granulation was dried for 1.5 hoursand stopped when the product temperature reached 36 C. The 3rd(batch017-10006) and 4th (batch 017-10007) capsule prototypes will usePROSOLV HD90, which is a higher density material with superior flowproperties and higher moisture content of 5.5% than the PROSOLV LM90.The moisture content of the PROSOLV HD90 is readily removed by fluid beddrying. The density of PROSOLV HD90 is about 0.55 g/mL. The PRUVlubricant will be removed for these batches.

Example 7: Wet Granulation Stability

SP-304 was extracted from the capsules by sonication at either at roomtemperature (RT) or cold temperature and the amount of peptide wasdetermined by HPLC. Initial percentages are based on the amount statedon the label.

Batch % peptide (initial) % peptide (1 mos at RT) 017-10006 101.1(sonicated RT)  97.6 (sonicated cold) 017-10008  97.5 (sonicated RT)108.2 (sonciated cold)

Example 8: 1M Capsule Stability in HDPE Bottles

Capsules contained 0.3 mg SP-304 with the remainder of the fill weight(up to 5 mg) made up by mannitol (Perlitol 300 DC). Each capsulecontained 1.5% by weight SP-304 and 98.5% mannitol. The capsule shellwas composed of HPMC. Amounts are relative to the amount specified onthe label (i.e., 0.30 mg peptide). The indicated number of capsules wasplaced in a high density polyethylene bottle with an induction seal andmolecular sieve desiccant for 1 month at either 2-8 C (first twocolumns) or 25 C and 60% relative humidity (last two columns). Theinitial amount of peptide present was 101% of the label claim. The lastrow gives the amount of peptide remaining after 1 month storage at theindicated temperature as determined by HPLC.

2-8 C. 2-8 C. 25 C./60RH 25 C./60RH 1-capsule per 6-capsules per1-capsule per 6-capsules per bottle bottle bottle bottle 100% 92% 92%98%

Example 9: Composition of Batch 1528-2855-RD (Capsules) and SprayCoating and Drying Process

Amount per Concentration % Item No. Ingredient unit (mg) w/w 1 SP-3040.3246 0.3246 2 Microcrystalline 99.10 99.10 cellulose (CelphereSCP-100) 3 Calcium chloride 0.2622 0.2622 dihydrate 4 Leucine USP 0.11710.1171 5 Hypromellose 0.2000 0.2000 (Methocel E5 PremLV) 6 PurifiedWater, USP 7.2 mL* n/a Total 100 100 *The amount of water is calculatedbased on use of 119.0 mL purified water for the whole batch containing5.356 g SP-304.

The spray drying process of making the batch 2855-RD is described below.

Preparation of Coating Dispersion:

Purified water was added to a glass container and stirred such that aliquid vortex was produced without introducing air. Then calciumchloride dihydrate was slowly added into the water. The mixture wasstirred until the salt was dissolved or well dispersed. Next, leucinewas slowly added and the resulting mixture was stirred until the aminoacid was dissolved or well dispersed. Afterward, methocel was slowlyadded and the mixture was stirred until methocel was completelydissolved. The solution could be warmed up to dissolve methocel, ifnecessary. The resulting excipient solution was allowed to cool to roomtemperature and pass through 80 mesh screen. Then, 127.9 g of screenedexcipient solution was added to a glass container and placed in an icebath for 0.5 to 1 hour until the solution reached 0° C. Next, SP-304 wasadded into the cold excipient solution. The mixture was stir vigorouslyto allow the peptide to dissolve in the cold solution. The resultingpeptide solution was kept cold in the ice bath as a spraying/coatingsolution.

Drug Layering

A Glatt GPCG-2 fluid bed processor (with top spray tower) with a Wursterinsert was set up for drug layering onto Celphere SCP-100 beads. Afterloading the Wurster column with Celphere SCP-100 beads, bed temperaturewas raised to 35° C. and maintained for 30 minutes with minimumfluidization of the beads. The bed temperature was reduced until anexhaust temperature of 35° C. was achieved. The pump tubing of theperistaltic pump used was primed by circulating the spraying solutionmentioned above. After the spraying apparatus was adjusted to obtain asatisfactory spray pattern, the coating solution was sprayed ontoCelphere SCP-100 beads until all coating solution was sprayed. Operatingparameters were recorded. The bed temperature and fluidization weremaintained until the beads were sufficiently dry. The fluidization wasthen reduced while the bed temperature was maintained at 35° C. for 10minutes. 2 g of beads were sampled for moisture analysis when the bedtemperature was kept at 35° C. When the moisture of the sampled beadsreached <5% moisture, the coated beads were discharged and loaded into adry container. LOD (loss on drying) 2.399%.

Example 10: Composition of Batch 1528-2851-RD (Tablets) and SprayCoating and Drying Process

Amount per Concentration % Item No. Ingredient unit (mg) w/w 1 SP-3040.3246 0.3607 2 Microcrystalline 88.88 98.75 cellulose (Avicel PH 102) 3Calcium chloride 0.2622 0.2913 dihydrate 4 Leucine USP 0.1171 0.1301 5Hypromellose 0.2000 0.2222 (Methocel E5 PremLV) 6 Magnesium stearate0.225 0.2500 7 Purified Water, USP 7.2 mL* n/a Total 90.0 100 *Theamount of water is calculated based on use of 119.0 mL purified waterfor the whole batch containing 5.356 g SP-304.

The spray coating and drying process of making the batch 2851-RD isdescribed below.

Preparation of Coating Dispersion:

Purified water was added to a glass container and stirred such that aliquid vortex was produced without introducing air. Then calciumchloride dihydrate was slowly added into the water. The mixture wasstirred until the salt was dissolved or well dispersed. Next, leucinewas slowly added and the resulting mixture was stirred until the aminoacid was dissolved or well dispersed. Afterward, methocel was slowlyadded and the mixture was stirred until methocel was completelydissolved. The solution could be warmed up to dissolve methocel, ifnecessary. The resulting excipient solution was allowed to cool to roomtemperature and pass through 80 mesh screen. Then, 127.9 g of screenedexcipient solution was added to a glass container and placed in an icebath for 0.5 to 1 hour until the solution reached 0° C. Next, SP-304 wasadded into the cold excipient solution. The mixture was stir vigorouslyto allow the peptide to dissolve in the cold solution. The resultingpeptide solution was kept cold in the ice bath as a spraying/coatingsolution.

Drug Layering

A Glatt GPCG-2 fluid bed processor (with top spray tower) with a Wursterinsert was set up for drug layering onto Avicel PH 102 beads. Afterloading the Wurster column with Avicel PH 102 beads, temperature wasraised to 35° C. and maintained for 30 minutes with minimum fluidizationof the beads. The bed temperature was reduced until an exhausttemperature of 35° C. was achieved. The pump tubing of the peristalticpump used was primed by circulating the spraying solution mentionedabove. After the spraying apparatus was adjusted to obtain asatisfactory spray pattern, the coating solution was sprayed onto AvicelPH 102 beads until all coating solution was sprayed. Operatingparameters were recorded. The bed temperature and fluidization weremaintained until the beads were sufficiently dry. The fluidization wasthen reduced while the bed temperature was maintained at 35° C. for 10minutes. 2 g of beads were sampled for moisture analysis when the bedtemperature was kept at 35° C. When the moisture of the sampled beadsreached <5% moisture, the coated beads were discharged and loaded into adry container. LOD (loss on drying)<5%.

The net weight of the coated blend was determined for calculation of theamount of magnesium stearate needed to lubricate the blend. Then themagnesium stearate was added to the coated blend and the mixture wasblended for 1 minute.

Compression

A Fette tablet press was set up. Then the blend mixture was loaded intothe powder hopper and tooling was installed. The weight of each tabletwas set to be 90 mg±5% and hardness to be 4-6 Kp. The weight, hardnessand thickness of tablets were measured and recorded every 5 to 10minutes. Friability measurement was also performed to ensuresatisfactory product.

Example 11: Composition of Batch 1528-2850-RD (Capsules) and Process

Concentration % Item No. Ingredient w/w 1 SP-304 0.3246 2Microcrystalline 99.43 cellulose (Avicel PH 102) 3 Magnesium stearate0.2500 4 HPMC capsule shells n/a Total 100

The dry blend process of making the batch 2850-RD is described below.

Blending:

Avicel PH 102 was screened through a 60 mesh screen. V-blenders (1 Qt,4Qt, and 16 Qt) were then dusted by the screened Avicel PH 102. SP-304was screened through a 200 mesh screen and loaded into the 1-QtV-blender. Then, about 80 g Avicel PH 102 was added into the 1-Qtblender and the mixture was blended for 10 minutes at 25 rpm. Themixture was then transferred to the 4-Qt V-blender which was pre-dustedby the screened Avicel PH 102. The 1-Qt blender was rinsed with Aviceland the rinse material was transferred to the 4-Qt blender. The rinsingwas repeated until all SP-304 was transferred to the 4-Qt blender. About200 g Avicel was added to the 4-Qt V-blender and the mixture was blendedfor 10 minutes. The resulting blend was then screened through a 60 meshscreen and then transferred into the pre-dusted 16-Qt blender (dustedwith 1500 g Avicel). The 4-Qt blender was rinsed with Avicel and therinse material was transferred to the 16-Qt blender. The remainingAvicel was added to the 16-Qt blender and the mixture was blended for 10minutes. The resulting blend was passed through Comil and then returnedto the 16-Qt blender and was further blended for 5 minutes. Properamount of magnesium stearate was weighed, screened through a 60 meshscreen, and added into the 16-Qt blender. The resulting mixture wasblended for 2 minutes.

Encapsulation

A MG2 Planeta capsule filler was set up. Average weight of the emptycapsule shells was determined and target capsule fill weight wascalculated (±5%). The blend from the above process was added into thehopper of the capsule filler and encapsulation was started. Run weightparameters were manually adjusted. Resulting capsules were then sortedaccording to the target fill weight.

Example 12: Composition of Batch 1528-2850B-RD (Tablets) and Process

Concentration % Item No. Ingredient w/w 1 SP-304 0.3246 2Microcrystalline 99.43 cellulose (Avicel PH 102) 3 Magnesium stearate0.2500 Total 100

The dry blend process of making the batch 2850B-RD is described below.

Blending:

Avicel PH 102 was screened through a 60 mesh screen. V-blenders (1 Qt,4Qt, and 16 Qt) were then dusted by the screened Avicel PH 102. SP-304was screened through a 200 mesh screen and loaded into the 1-QtV-blender. Then, about 80 g Avicel PH 102 was added into the 1-Qtblender and the mixture was blended for 10 minutes at 25 rpm. Themixture was then transferred to the 4-Qt V-blender which was pre-dustedby the screened Avicel PH 102. The 1-Qt blender was rinsed with Aviceland the rinse material was transferred to the 4-Qt blender. The rinsingwas repeated until all SP-304 was transferred to the 4-Qt blender. About200 g Avicel was added to 4-Qt V-blender and the mixture was blended for10 minutes. The resulting blend was then screened through a 60 meshscreen and then transferred into the pre-dusted 16-Qt blender (dustedwith 1500 g Avicel). The 4-Qt blender was rinsed with Avicel and therinse material was transferred to the 16-Qt blender. The remainingAvicel was added to the 16-Qt blender and the mixture was blended for 10minutes. The resulting blend was passed through Comil and then returnedto the 16-Qt blender and was further blended for 5 minutes. Properamount of magnesium stearate was weighed, screened through a 60 meshscreen, and added into the 16-Qt blender. The resulting mixture wasblended for 2 minutes.

Compression

A Fette tablet press was set up. Then the blend mixture was loaded intothe powder hopper and tooling was installed. The weight of each tabletwas set to be 90 mg±5% and hardness to be 4-6 Kp. The weight, hardness,and thickness of tablets were measured and recorded every 5 to 10minutes. Friability measurement was also performed to ensuresatisfactory product.

Example 13: Composition of Dry Blend Tablet Formulation 1528-3161-RD, 1mg for Vacuum Drying

Concentration % Item No. Ingredient w/w 1 SP-304 1.176 2Microcrystalline 98.57 cellulose (Avicel PH 102) 3 Magnesium stearate0.2500 Total 100

Example 14: Composition of Dry Blend Tablet Formulation 1528-3162-RD, 1mg with Low-Moisture Cellulose

Concentration % Item No. Ingredient w/w 1 SP-304 1.176 2Microcrystalline 97.09 cellulose (Avicel PH 112) 3 Magnesium stearate0.2500 Total 100

Example 15: Composition of Spray Coated Trehalose Granules TabletFormulation 1528-3170-RD, 1 mg

Concentration % Item No. Ingredient w/w 1 SP-304 1.176 2 Trehalosegranules 70.48 3 Methocel ES Premium 0.50 LV 4 Histidine (in coating0.9225 solution) 5 Calcium ascorbate 0.100 6 Purified water N/A 7Trehalose powder (in 1.0176 coating solution) 8 Microcrystalline 25.00cellulose (Avicel PH 200) 9 Histidine 0.5535 10 Magnesium stearate0.2500 Total 100

The process for making spray coated trehalose Granules tabletformulation 1528-3170-RD is described below.

Preparation of the Coating Dispersion

Add purified water to labeled container and begin stirring. Stir suchthat a liquid vortex is produced without introducing air into liquid.Slowly add Methocel to solution. Stir until methocel is completelydissolved. Warm the solution if necessary to dissolve Methocel (≦50°C.). Solution must be cooled before adding other materials. AddTrehalose to solution. Stir until materials are dissolved. Add CalciumAscorbate to solution. Stir until materials are dissolved. Adjust pH to7.0 with 1N NaOH solution if pH >7.0. Record adjusted pH. Place theCoating Solution in an ice bath and allow it stay in the batch for 0.5to 1 hour until it reaches the ice temperature. Check with a thermometerto ensure at ice temperature. Weigh portions of required amount of APIon a weighing boat and add each portion carefully to the cold ExcipientSolution. Stir vigorously to allow peptide wetting and dissolving in thecold solution. Total amount of peptide must equal 14.107 g. Continuestirring solution such that a liquid vortex is produced withoutintroducing air into liquid. Stir until PLECANATIDE is completelydissolved. Keep peptide solution cold all the time in the ice bath. AddHistidine to solution. Stir not more than 10 min to dissolve thematerial. Obtain final pH of the Coating Solution. Obtain net weight ofthe Coating Solution. Coating Solution must be used within 30 min toavoid coloration.

Drug Layering

Setup Glatt GPCG2 with Wurster insert according to SOP EQP-OCM-064 fordrug layering onto Trehalose Granules with coating dispersion. Use GlattGPCG2 In-process form, “EQP-OCM-064-F1,” to record in-processinformation. Turn unit on and preheat column. Fluid Bed Processor: GlattGPCG-2. Filter: 200 micron screen. Product Container: 4″ wurster,stainless steel. Insert height from bottom: 1″. Spray direction: TopSpray. Fluid Nozzle Size/Type: 1 mm. Pump: Peristaltic, Master Flex LS.Tubing: Nalge #14 Silicon. Bed Temperature: ≦40° C. Inlet airtemperature: Adjust to meet bed temperature target. Outlet airtemperature: Monitor & record. Spray rate: initial rate 4-6 g/min,adjust as required. Atomizing air pressure: 20 psi. Air flow: 60 cmh andadjust for fluidization. Prepare double polyethylene bags large enoughto hold drug layered Granules. Load column with Trehalose. Increase bedtemperature to 35° C. and maintain for 30 minutes with minimumfluidization of the Granules. Reduce bed temperature until an exhausttemperature of 35° C. is achieved. Prime pump tubing by circulatingspraying solution; must not use more than 40 g for tubing priming.Adjust the spraying apparatus to obtain satisfactory spray pattern.Coating Solution Weight after priming should >317 g. Record initialweight below before spraying onto trehalose. Start spraying the coatingsolution onto Trehalose Granules. Record operating parameters on fluidbed processing form. Stop spraying when 297.2 g of coating solution hasbeen sprayed. Maintain bed temperature and continue fluidization untilGranules are sufficiently dry. Reduce fluidization and maintain bedtemperature at 35° C. for 10 minutes. Do not cool down the Granules.Sample 2 g for moisture analysis until moisture is below 1%. Dischargecoated Granules into pre-prepared and labeled container (with tareweight) lined with double polyethylene bag. Calculate net weight of druglayered Granules. Setup Lyophilizer per SOP EQP-OCM-00002. Load druglayered granules into a Lyoguard tray (Save bags). Use recipe 3 to dryblend overnight. Discharge dried blend into saved polyethylene bags.Obtain final moisture of the dried granules. Record final Moisture(<1%). Calculate net weight of dried Granules.

Blending

Screen required Avicel and pass through 60 mesh screen. Setup 4 qtV-blender per SOP EQP-OCM-00056. Weigh amount of Histidine needed andblend with small amount of Avicel weighed. Charge into 4 qt. V-blender.Transfer Plecanatide Dried Granules into the V-Blender. Rinse 2-3 timesthe Lyoguard tray from Step 24 with adequate amount of Weighed Avicel.Transfer rinses into 4 qt. V-b; ender. Transfer all remainingPre-weighed/screened Avicel into the V-Blender. Mix for 15 minutes.Weigh and screen Magnesium Stearate through a 60 mesh screen. ChargeMagnesium Stearate to the 4 qt V-Blender. Ensure the cover is securelyclosed with no potential powder leakage during blending. Blend for 2minutes.

Compression

Set-up Korsch press per SOP EQP-OCM-00087. Install 0.250″ StandardConcave Round Plain tolling. Obtain blend Assay results and calculateTarget Tablet Weight. Acceptable weight range of tablets is ±5.0%. Loadthe Final Blend into the powder hopper. Refill as necessary. Adjust fillweight to obtain tablets in the range of 95.0-105.0 mg and hardness inthe range of 4-6 kP. Verify friability is NMT 1.0%. Check 5 tabletweights periodically every 5-10 min to ensure tablet weight is withinthe range and record on form QRA-DOC-00011-F6. After tablet weights arerecorded, obtain and record 3 tablet hardness and thickness during theperiodic weight check. Continue to compress acceptable tablets until theblend is used up. Once press is running properly to achievespecifications above, perform final Friability test and record results(Spec: NMT 1.0%).

Example 16: Composition of Spray Coated Trehalose Granules TabletFormulation 1528-3171-RD, 1 mg

Concentration % Item No. Ingredient w/w 1 SP-304 1.167 2 Trehalosegranules 70.31 3 Methocel ES Premium 0.50 LV 4 Arginine 1.657 5 Calciumascorbate 0.100 6 Water for injection N/A 7 Trehalose powder (in 1.0176coating solution) 8 Microcrystalline 25.00 cellulose (Avicel PH 200) 9Magnesium stearate 0.2500 Total 100

The process for making spray coated trehalose Granules tabletformulation 1528-3171-RD is described below.

Preparation of Coating Solution

Add purified water (Item 6) to labeled container and begin stirring.Stir such that a liquid vortex is produced without introducing air intoliquid. Slowly add Methocel to solution. Stir until methocel iscompletely dissolved. Warm the solution if necessary to dissolveMethocel (≦50° C.). Record appearance of solution. Solution must becooled before adding other materials. Add Trehalose to solution. Stiruntil materials are dissolved. Record appearance of solution. AddArginine to solution. Stir until materials are dissolved. Recordappearance of solution. Add Calcium Ascorbate to solution. Stir untilmaterials are dissolved. Record appearance of solution. Adjust solutionpH to pH 8.5-8.6 with concentrated HCl followed by adjust pH to 8.3-8.4with 10N HCl. Record final adjusted pH. Place the Coating Solution in anice bath and allow it stay in the batch for 0.5 to 1 hour until itreaches the ice temperature. Check with a thermometer to ensure at icetemperature. Weigh portions of required amount of API on a weighing boatand add each portion carefully to the cold Excipient Solution. Stirvigorously to allow peptide wetting and dissolving in the cold solution.Total amount of peptide must equal 14.006 g. Continue stirring solutionsuch that a liquid vortex is produced without introducing air intoliquid. Stir until PLECANATIDE is completely dissolved. Keep peptidesolution cold all the time in the ice bath. Weigh 5.0 g of WFI to rinseAPI container. Carefully rinse the side of coating solution containerand completely transfer the rinse back to the coating solutioncontainer. Obtain final pH of the Coating Solution. Obtain net weight ofthe Coating Solution (˜360.3 g). Coating Solution must be used within assoon as possible.

Drug Layering

Setup Glatt GPCG2 with Wurster insert according to SOP EQP-OCM-064 fordrug layering onto Trehalose Granules with coating dispersion. Use GlattGPCG2 In-process form, “EQP-OCM-064-F1,” to record in-processinformation. Turn unit on and preheat column.

Fluid Bed Processor: Glatt GPCG-2. Filter: 200 micron screen. ProductContainer: 4″ wurster, stainless steel. Insert height from bottom: 1″.Spray direction: Top Spray. Fluid Nozzle Size/Type: 1 mm. Pump:Peristaltic, Master Flex LS. Tubing: Nalge #14 Silicon. Bed Temperature:≦40° C. Inlet air temperature: Adjust to meet bed temperature target.Outlet air temperature: Monitor & record. Spray rate: initial rate 4-6g/min, adjust as required. Atomizing air pressure: 20 psi. Air flow: 60cmh and adjust for fluidization. Load column with Trehalose G. Increasebed temperature to 35° C. and maintain for 30 minutes with minimumfluidization of the Granules. Reduce bed temperature until an exhausttemperature of 35° C. is achieved. Prime pump tubing with coatingsolution. Must not use more than 40 g for tubing priming. Adjust thespraying apparatus to obtain satisfactory spray pattern. Record initialweight below before spraying onto trehalose. Start spraying the coatingsolution onto Trehalose Granules. Record operating parameters on fluidbed processing form. Stop spraying when 300.3 g of coating solution hasbeen sprayed. Maintain bed temperature and continue fluidization untilGranules are sufficiently dry. Reduce fluidization and maintain bedtemperature at 35° C. for 10 minutes. Do not cool down the Granules.Sample 2 g for moisture analysis until moisture is below 1%. Dischargecoated Granules into pre-prepared and labeled container (with tareweight) lined with double polyethylene bag. Calculate net weight of druglayered Granules. If moisture is >1%, vacuum dry blend as follows: SetupLyophilizer per SOP EQP-OCM-00002. Load drug layered granules into aLyoguard tray. Use recipe 3 to dry blend overnight. Discharge driedblend into saved polyethylene bags. Obtain final moisture of the driedgranules. Calculate net weight of dried Granules.

Blending

Screen required Avicel and pass through 60 mesh screen. Setup 4 qtV-blender. Transfer Plecanatide Dried Granules into the V-Blender. Savebag for discharging final blend. Rinse 2-3 times the Lyoguard tray andbag with adequate amount of Weighed Avicel. Transfer rinses into 4 qt.V-b; ender. Transfer all remaining Pre-weighed/screened Avicel into theV-Blender. Mix for 20 minutes. Weigh and screen Magnesium Stearatethrough a 60 mesh screen. Charge Magnesium Stearate to the 4 qtV-Blender. Ensure the cover is securely closed with no potential powderleakage during blending. Blend for 2 minutes. Sample 3×350 mg of blendat three locations. Obtain exact weight of each sample that has beentransferred into the sampling bottle.

Compression

Set-up Korsch press per SOP EQP-OCM-00087. Install 0.250″ StandardConcave Round Plain tolling. Obtain blend Assay results and calculateTarget Tablet Weight. Acceptable weight range of tablets is ±5.0%. Loadthe Final Blend into the powder hopper. Refill as necessary. Adjust fillweight to obtain tablets in the range of 95.0-105.0 mg and hardness inthe range of 4-6 kP. Verify friability is NMT 1.0%. Check 5 tabletweights periodically every 5-10 min to ensure tablet weight is withinthe range. After tablet weights are recorded, obtain and record 3 tablethardness and thickness during the periodic weight check. Continue tocompress acceptable tablets until the blend is used up. Once press isrunning properly to achieve specifications above, perform finalFriability test and record results (Spec: NMT 1.0%).

Example 17: Composition of Spray Coated Trehalose Granules TabletFormulation 1528-3172, 1 mg

Concentration % Item No. Ingredient w/w 1 SP-304 1.167 2 Trehalosegranules 70.81 3 Methocel ES Premium 0.50 LV 4 TRIS 1.1524 5 Calciumascorbate 0.100 6 Water for injection N/A 7 Trehalose powder (in 1.0176coating solution) 8 Microcrystalline 25.00 cellulose (Avicel PH 200) 9Magnesium stearate 0.2500 Total 100

The process for making spray coated trehalose granules tabletformulation 1528-3172-RD is described below.

Preparation of Coating Solution

Add purified water to labeled container and begin stirring. Stir suchthat a liquid vortex is produced without introducing air into liquid.Slowly add Methocel to solution. Stir until methocel is completelydissolved. Warm the solution if necessary to dissolve Methocel (≦50°C.). Record appearance of solution.

Solution must be cooled before adding other materials. Add Trehalose tosolution. Stir until materials are dissolved. Record appearance ofsolution. Add TRIS to solution. Stir until materials are dissolved.Record appearance of solution. Add Calcium Ascorbate to solution. Stiruntil materials are dissolved. Record appearance of solution. Obtainsolution pH: Adjust pH to pH 7.8-7.9 with concentrated HCl followed byadjust pH to 7.7-7.6 with 10N HCl. Record final adjusted pH. Place theCoating Solution in an ice bath and allow it stay in the batch for 0.5to 1 hour until it reaches the ice temperature. Check with a thermometerto ensure at ice temperature. Weigh portions of required amount of APIon a weighing boat and add each portion carefully to the cold ExcipientSolution. Stir vigorously to allow peptide wetting and dissolving in thecold solution. Total amount of peptide must equal 14.006 g. Continuestirring solution such that a liquid vortex is produced withoutintroducing air into liquid. Stir until PLECANATIDE is completelydissolved. Keep peptide solution cold all the time in the ice bath.Weigh 5.0 g of WFI to rinse API container. Carefully rinse the side ofcoating solution container and completely transfer the rinse back to thecoating solution container. Obtain final pH of the Coating Solution.Obtain net weight of the Coating Solution (354.2 g). Coating Solutionmust be used as soon as possible.

The blending and compression processes for batch 1528-3172-RD aresimilar to that described above for batch 1528-3171-RD.

Example 18: Composition of 1 mg Dry Blend Tablet Formulation1528-2925-RD

Concentration % Item No. Ingredient w/w 1 SP-304 1.106 2Microcrystalline 98.64 cellulose (Avicel PH 102) 3 Magnesium stearate0.2500 Total 100

Example 19: Composition of 3 mg Dry Blend Tablet Formulation1528-2926-RD

Concentration % Item No. Ingredient w/w 1 SP-304 3.318 2Microcrystalline 96.43 cellulose (Avicel PH 102) 3 Magnesium stearate0.2500 Total 100

Other batches were prepared by the processes similar to those describedin Examples 9-12. Their compositions are listed below.

Batch 500-55: 0.33% plecanatide, 95.17% microcyrstalline cellulose, 4.0%sodium starch glycolate, and 0.5% magnesium stearate.

Batches 1528-2907-RD and 2010F100A: 3.318% plecanatide, 96.43% Avicel,and 0.25% Mg stearate.

Batches 1528-2906-RD and 2010F099A: 1.106% plecanatide, 98.65% Avicel,and 0.25% Mg stearate.

Batches 1528-2890-RD and 2010F101A: 0.3246% plecanatide, 99.43% Avicel,and 0.25% Mg stearate.

Formula compositions for batches 11H141, 11H152, and 11H140 in thistable below (not previously disclosed) are the same as the formulacompositions for GMP stability batches 2010F101A, 2010F099A, and2010F100A, respectively.

Example 20: Plecanatide Tablet and Capsule Stability

Capsules and tablets of different batches were tested for theirstability and the results were provided. Unless otherwise specified, 1M,2M, 3M, or 4M in the tables below denotes that the measurements werecarried out at the end of 1, 2, 3, or 4 month(s) of the storage period.

Potency Summary:

This test was performed by taking a composite sample of about 5 units todetermine the average potency of the sample. The table below shows thestability of capsules or tablets in terms of potency (% of label claim).

Potency (% Label Claim) Storage Condition 40 C./ 30 C./ 75 RH 65 RH 25C./60 RH 5 C. Lot Package 1 2 3 1 2 3 1 2 3 7 10 1 2 3 4 7 8.5(description) Bulk* Package Initial M M M M M M M M M M M M M M M M M1528-2850- 88 HDPE bottle 89 87 89 91 80 89.3 89 RD (0.3 mg Oxyguard 9191 92 91 79 88.9 90 dry blend bottle capsules) Blister strip 90 90 85 8891 79 90 1528-2855- 94 HDPE bottle 101 100 96 102 88 98 RD (0.3 mgOxyguard 101 96 99 104 87 100 coated blend bottle capsule) Blister strip97 103 99 98 87 97 500-55 97 HDPE bottle 97 94 95 96 84 98 (0.3 mg dryOxyguard 98 96 96 102 83 97 blend bottle capsule) Blister strip 93 97 9395 106 83 96 1528-2850B- 76 HDPE bottle 85 88 94 83 67 70 RD (0.3 mgOxyguard 84 84 88 74 74 80 dry blend bottle tablet) 1528-2851- 96 HDPEbottle 115 72 90 99 99 78 RD (0.3 mg Oxyguard 81 88 83 111 85 96 coatedbottle particle tablet) 2010F100A 101 Blister strip 97 95 94 91 95 95 9297 95 93 97 94 94 (3 mg dry blend capsule) 2010F101A 97 Blister strip 9291 91 86 94 92 85 95 93 88 95 95 92 (0.3 mg dry blend capsule) 2010F099A98 Blister srtip 94 92 91 89 93 94 89 94 94 91 95 94 92 (1 mg dry blendcapsule) 11H141 103 Blister strip 101 95 92 87 98 93 92 96 92 95 100 9797 (0.3 mg dry blend capsule) 11H152 102 Blister strip 97 91 91 93 94 9596 96 95 96 97 95 97 (1 mg dry blend capsule) 11H140 105 Blister strip99 94 95 94 95 94 97 99 95 97 99 97 97 (3 mg dry blend capsule)1528-2925- 99 Oxyguard 99 103 RD (1 mg 40 cc with dry blend PhamaKeeptablet) 1528-2926- 100 Oxyguard 94 93 RD (3 mg 40 cc with dry blendPhamaKeep tablet) 1528-2907- 98 RD (3 mg dry blend capsule) 1528-2906-98 RD (1 mg dry blend capsule) 1528-2890- 93 RD (0.3 mg dry blendcapsule) *Blend

As demonstrated by the table above, there was little or no appreciableloss in potency after storage under accelerated conditions (40 C/75RH or30 C/65RH), which suggests that these capsules or tablets could bestable at room temperature for 18 months or for longer times ifrefrigerated or stored at 25 C.

Water content summary: The table below shows that the water content wasstable over the testing period in the packages evaluated for variouscapsule/tablet compositions. This further demonstrated that productswere stable.

Water packaged product Water 40 C./ 30 C./ (in- 75 RH 65 RH 25 C./60 RH5 C. proc- 1 2 3 1 2 3 1 2 3 7 10 1 2 3 4 7 8.5 Lot ess) PackagingInitial M M M M M M M M M M M M M M M M M 1528-2850- 32-count, 5.03 5.643.00 2.22 2.39 5.48 1.8 RD 0.3 mg HDPE bottle, dry blend 60 cc, N2,capsule 2 g mol. sieve 32-count, 5.07 5.24 4.28 5.33 4.08 5.31 3.7Oxyguard bottle, 40 cc, PharmaKeep KD-20 Blister N2 4.21 4.87 5.80 4.764.31 4.09 2.8 1528-2855- 2.40 32-count, 0.57 0.47 1.63 0.68 0.42 0.2 RD0.3 mg HDPE bottle, coated bead 60 cc, N2, capsule 2 g mol. sieve32-count, 2.10 1.05 1.29 2.07 0.30 0.8 Oxyguard bottle, 40 cc,PharmaKeep KD-20 Blister strip 0.73 2.11 0.54 0.58 0.32 0.3 500-55 HDPEbottle 5.63 4.19 5.51 5.79 2.98 2.7 0.3 mg Oxyguard 5.78 4.69 5.90 5.662.99 2.8 dry blend bottle capsule Blister strip 4.09 5.78 4.17 5.53 6.163.12 2.9 1528- 32-count, 4.09 4.03 6.28 6.10 2.86 2.1 2850B-RD HDPEbottle, 0.3 mg dry 60 cc, N2, blend tablet 2 g mol. sieve 32-count, 4.814.91 6.15 6.30 4.05 3.4 Oxyguard bottle, 40 cc, PharmaKeep KD-201528-2851- 3.32 32-count, 4.33 4.50 5.09 5.90 2.55 1.5 RD 0.3 mg HDPEbottle, coated 60 cc, N2, particle 2 g mol. tablet sieve 32-count, 5.154.88 5.82 6.02 4.34 3.0 Oxyguard bottle, 40 cc, PharmaKeep KD-202010F100A Blister strip 4.7  4.5  4.6 4.4  4.5 4.7 4.4  4.5 4.8 4.4  4.54.8 4.5 (3 mg dry blend capsule) 2010F101A Blister strip 4.5  4.8  4.74.7  4.5 4.7 4.3  4.4 4.7 4.3  4.5 4.7 4.2 (0.3 mg dry blend capsule)2010F099A Blister strip 4.6  4.4  4.6 4.4  4.5 4.5 4.3  4.4 4.6 4.4  4.24.7 4.3 (1 mg dry blend capsule) 11H141 Blister strip 5   4.8  4.9 4.9 5.1 4.9 4.8  5.0 5.0 4.9  5.0 4.9 4.9 (0.3 mg dry blend capsule) 11H152Blister strip 5.2  4.8  4.9 4.8  4.8 4.8 4.9  4.8 4.8 4.9  5.0 4.9 4.8(1 mg dry blend capsule) 11H140 Blister strip 5.2  5.0  5.0 5.0  4.9 5.05.0  4.9 5.0 4.9  4.9 4.9 4.8 (3 mg dry blend capsule) 1528-2925-Oxyguard 4.9 4.0 RD (1 mg 40 cc with dry blend PharnaKeep tablet)1528-2926- Oxyguard 4.0 4.0 RD (3 mg 40 cc with dry blend PharnaKeepcapsule) 1528-2907- Bulk 4.78 RD 3 mg capsule dry blend capsule1528-2906- Bulk 4.84 RD 1 m capsule dry blend capsule 1528-2890- Bulk4.8  RD capsule

Impurity Summary:

The table below shows the product stability in terms of HPLC or UPLC oftotal impurities as a function of time and storage condition. The datain the table suggest that the increase in total impurities in testedbatches except batch 500-55 be no greater than 7% at room temperatureafter 18 months. It also suggest that the increase in total impuritiesin all tested 1528-2855-RD batche in different packages be no greaterthan 7% at 30° C. for 18 months. It was also observed that the1528-2855-RD batch had less impurity increase than the 1528-2850-RDbatch or was more stable than the 1528-2850-RD batch.

Total impurities % area 40 C./ 30 C./ 75 RH 65 RH 25 C./60 RH 5 C. 1 2 31 2 3 1 2 3 7 10 1 2 3 4 7 8.5 Batch Package Initial M M M M M M M M M MM M M M M M M 1528-2850- HDPE bottle 3.2  5.1 5.9 4.4 3.8 4.8 3.1 3.7 RDOxyguard 5.7 7.4 5.3 4.3 5.3 3.1 3.5 bottle Blister strip 5.5 7.0 5.04.3 5.5 3.7 1528-2855- HDPE bottle 3.5  3.6 5.1 3.8 3.4 4.4 3.4 RDOxyguard 3.9 4.4 4.1 3.7 4.0 3.7 bottle Blister strip 4.0 5.2 4.0 3.64.2 3.8 500-55 HDPE bottle 3.2  5.7 8.4 5.4 4.4 6.0 3.5 Oxyguard 5.6 7.05.1 4.3 5.6 3.5 bottle Blister strip 6.5 8.0 5.7 4.8 6.5 3.6 1528- HDPEbottle 3.6  5.0 6.5 4.5 3.9 4.7 3.7 2850B-RD Oxyguard 5.6 7.3 4.7 4.14.9 3.6 bottle 1528-2851- HDPE bottle 3.7  4.2 5.1 4.0 3.8 3.9 3.7 RDOxyguard 4.9 6.8 4.7 4.4 4.3 3.9 bottle 2010F101A Blister strip 2.1  4.43.9  4.7 2.9 3.2 3.4 3.1 2.7 3.2 2.0 1.3 2.0 (0.3 mg dry blend capsule)2010F099A Blister strip 2.9  3.7 3.8  4.3 3.1 3.1 3.6 2.7 2.9 3.2 2.42.4 2.4 (1 mg dry blend capsule) 2010F100A Blister strip 2.4  3.2 3.6 4.2 2.8 2.8 3.0 2.6 2.7 2.9 2.4 2.5 2.7 (3 mg dry blend capsule) 11H141Blister strip 1.3  3.3 4.2  4.5 2.5 3.6 3.3 2.0 2.8 2.9 1.4 1.5 1.8 (0.3mg dry blend capsule) 11H152 Blister strip (1 mg dry 2.4  3.6 4.2  4.12.6 3.2 3.1 2.6 3.1 2.9 2.3 2.3 2.1 blend capsule) 11H140 Blister strip2.1  3.5 3.7  4.5 2.6 2.7 3.3 2.5 2.7 2.9 2.3 2.2 1.8 (3 mg dry blendcapsule) 1528-2925- Oxyguard 2.7 1.7 RD (1 mg 40 cc with dry blendPharnaKeep tablet) 1528-2926- Oxyguard 2.6 RD (3 mg 40 cc with dry blendPharnaKeep capsule) 1528-2906- HDPE bottle 1.83 5.18 RD 1528-2907- HDPEbottle 1.85 4.58 RD 1528-2890- Bulk 1.9  RDContent uniformity: This test was performed by placing 10 individualcapsule/tablet units in 10 individual bottles and potency of each unitwas measured to show whether individual capsules or tablets have uniformpotency (% label claim or % LC).

0.3 mg Dry blend tablet 1528-2850B-RD % LC 1528-2850B- Sample RD (drytabs) 1 78.62 2 91.43 3 86.52 4 90.9 5 84.83 6 95.29 7 75.69 8 76.87 984.92 10  86.9 Mean 85.2 std. dev 6.51 % RSD 7.64

0.3 mg Coated particle tablet 1528-2851-RD Weight % Label Sample (mg)Claim 1 88.86 69.55 2 89 94.41 3 88.89 94.34 4 88.6 72.18 5 88.37 142.526 88.76 149.44 7 89.42 78.8 8 88.56 131.08 9 89.08 102.55 10  88.7899.13 Mean 103.4 St. Dev 28.53 % RSD 27.59

3 mg Dry blend 1 mg Dry blend 0.3 mg Dry blend capsule 1528- capsule1528-2906- capsule 1528-2890 2907-RD RD Sample % LC Sample % LC Sample %LC 1 87.2 1 94.5 1 98.1 2 94.6 2 101.2 2 101.8 3 92.6 3 97.9 3 93.1 494.2 4 94.5 4 97.5 5 93.5 5 95.9 5 97.9 6 91.7 6 95.2 6 97.1 7 91.6 796.1 7 94.5 8 99 8 99 8 100.1 9 91.8 9 93.8 9 98.1 10  92.1 10  93.4 10 97.9 Mean 92.8 Mean 96.2 Mean 97.6 RSD 3.20% RSD 2.60% RSD 2.50%AV(10)*** 12.8 AV(10) 8.4 AV(10) 6.8 ***AV = acceptance value used forUPS <905> content uniformity. Idealy AV should be less than 15 to passUSP <905> content uniformity.

0.3 mg dry blend capsule 1528-2850-RD Original Re-preparation Sample %LC % LC 1 82.73 85.87 2 84.57 89.45 3 80.29 91.39 4 84.88 88.45 5 85.286.96 6 82.9 84.84 7 84.75 86.21 8 86.58 91.37 9 84.34 88.79 10  88.8284.75 Mean 84.51 87.81 std. dev 2.288445 2.467121 % RSD 2.7 2.8

Conte1528- 1528- 2855-RD 2850B-RD Sample % LC Sample % LC 1 88.82 178.62 2 93.73 2 91.43 3 89.06 3 86.52 4 84.94 4 90.9 5 89.93 5 84.83 688.7 6 95.29 7 88.71 7 75.69 8 86.85 8 76.87 9 86.92 9 84.92 10  91.3310  86.9 Mean 88.9 Mean 85.2 std. dev 2.45 std. dev 6.51 % RSD 2.76 %RSD 7.64

500-55 Sample % label claim 1 96.90% 2 99.40% 3 103.20% 4 96.90% 5100.00% 6 99.60% 7 96.90% 8 102.80% 9 96.80% 10  93.90% Mean 98.60% SD2.91 RSD 3.00% AV 7.1 (PASS)

The data in the tables above show that all of the batches yield verygood content uniformity acceptable for commercial product.

Dissolution 50-Rpm Summary:

The tables below are summaries of the dissolution of drug from capsulesor tablets in an unconventional small-volume apparatus needed to measurethe small amount of drug in the units using slow stirring to look forchanges in dissolution over time. The test was performed by placing oneunit into a very small volume of water at 37 C with a paddle stirring at50-rpm (which is slow) and data were collected at 15, 30 45, and 60minutes to show the drug release rate over time. These tested productsare “immediate release” oral solid dosage forms and a conventionalrequirement is to have about 75% released in about 45 minutes. Thetables summarize the results at 45 minutes and indicate that dissolutionwas stable over time.

Dissolution (% label claim at 45 minutes) 40 C./ 30 C./ Lot Initial 75RH 65 RH 25 C. 5 C. (description) bulk 0 M 1 M 2 M 3 M 3 M 4 M1528-2850- Vessel 1 85 78 84 81 86 83 RD (dry Vessel 2 87 73 90 82 84 85blend V-Cap Vessel 3 88 79 85 79 91 87 capsule Vessel 4 84 86 87 78 8385 HDPE Vessel 5 89 72 89 80 79 90 bottle) Vessel 6 88 81 85 82 88 83Average 87 78 87 80 85 85 RSD 2 6.4 2.7 2.1 5.0 2.9 1528-2850- Vessel 185 69 89 79 88 82 RD (dry Vessel 2 87 75 89 87 81 85 blend VCap Vessel 388 77 87 86 84 86 capsule Vessel 4 84 80 87 83 83 80 OxyGuard Vessel 589 71 88 89 84 84 bottle) Vessel 6 88 76 88 79 86 89 Average 87 75 88 8484 84 RSD 2 5.3 1.2 5.2 3.1 3.6 1528-2850- Vessel 1 85 75 59 86 73 83 RD(dry Vessel 2 87 89 77 79 81 81 blend V-Cap Vessel 3 88 88 83 87 74 84capsule Vessel 4 84 89 67 93 85 83 blister strip) Vessel 5 89 93 75 8282 84 Vessel 6 88 90 82 90 67 87 Average 87 87 74 86 77 84 RSD 2 7 12.56.3 8.6 2.4

Dissolution (% label claim at 45 minutes) 40 C./ 30 C./ Lot Initial 75RH 65 RH 25 C. (description) bulk 1 M 2 M 3 M 3 M 1528-2855-RD Vessel 1104 85 100 79 83 (coated bead Vessel 2 89 90 97 83 88 V-Cap capsuleVessel 3 91 84 71 91 50 HDPE bottle) Vessel 4 88 64 73 94 88 Vessel 5 9475 72 75 92 Vessel 6 93 80 39 96 94 Average 93 80 75 86 83 RSD 6 12 299.7 20 1528-2855RD Vessel 1 104 88 80 87 78 (coated bead Vessel 2 89 7991 86 94 V-cap capsule Vessel 3 91 84 63 92 74 OxyGuard Vessel 4 88 9298 90 98 bottle) Vessel 5 94 89 81 81 93 Vessel 6 93 44 99 81 78 Average93 79 85 86 86 RSD 6 23 16 5.3 12.1 1528-2855-RD Vessel 1 104 85 98 10081 (coated bead Vessel 2 89 84 94 63 80 V-cap capsule Vessel 3 91 97 9682 87 blister strip) Vessel 4 88 94 96 55 74 Vessel 5 94 64 75 95 66Vessel 6 93 96 102 89 82 Average 93 87 93 81 78 RSD 6 14 10 22.4 9.2

Dissolution (% label claim at 45 minutes) Lot Initial 40 C./75RH 30C./65RH (description) bulk 1 M 2 M 3 M 1528-2851- Vessel 1 58% 67 68 89RD (coated Vessel 2 77% 84 78 124 particle tablet Vessel 3 57% 62 68 70HDPE bottle) Vessel 4 96% 110 84 105 Vessel 5 95% 65 107 61 Vessel 6 64%103 76 51 Average 74% 82 80 83 RSD 24% 26 18 33 1528-2851- Vessel 1 58%89 54 118 RD (coated Vessel 2 77% 73 101 69 particle tablet Vessel 3 57%75 82 80 OxyGuard Vessel 4 96% 68 67 73 bottle) Vessel 5 95% 76 162 96Vessel 6 64% 97 82 95 Average 74% 80 91 89 RSD 24% 14 42 21

Dissolution (% label claim at 45 minutes) Lot Initial 40 C./75 RH 30C./65 RH (description) bulk 1 M 2 M 3 M 1528-2850B- Vessel 1 90% 88 9692 RD (dry blend Vessel 2 69% 79 82 92 tablet HDPE Vessel 3 83% 76 10085 bottle) Vessel 4 94% 96 86 94 Vessel 5 88% 89 89 83 Vessel 6 92% 8397 83 Average 86% 85 92 88 RSD 11% 8.2 8 5.6 1528-2850B- Vessel 1 90% 7480 91 RD (dry blend Vessel 2 69% 97 87 95 tablet Vessel 3 83% 91 86 90OxyGuard Vessel 4 94% 94 91 90 bottle) Vessel 5 88% 83 91 89 Vessel 692% 91 76 84 Average 86% 88 85 90 RSD 11% 9.6 7 4.0

Dissolution (% label claim at 45 minutes) 40 C./ 30 C./ Lot Initial 75RH 65 RH 25 C. (description) bulk 0 M 1 M 2 M 3 M 3 M 500-55 (dry Vessel1 95 90 92 91 89 blend V-Cap Vessel 2 98 85 98 97 98 Plus capsule Vessel3 69 85 96 94 76 HDPE bottle) Vessel 4 94 89 95 100 97 Vessel 5 99 89 9798 86 Vessel 6 104 100 99 94 92 Average 93 89 96 96 90 RSD 13.1 6.2 2.43.6 9.1 500-55 (dry Vessel 1 95 84 103 99 94 blend V-Cap Vessel 2 98 97101 95 103 Plus capsule Vessel 3 69 97 99 98 97 OxyGuard Vessel 4 94 9297 92 96 bottle) Vessel 5 99 91 100 95 101 Vessel 6 104 96 95 93 91Average 93 93 99 95 97 RSD 13.1 5.3 2.7 2.7 4.3 500-55 (dry Vessel 1 9598 99 89 98 blend V-Cap Vessel 2 98 101 88 94 87 Plus capsule Vessel 369 107 90 89 96 foil blister) Vessel 4 94 96 90 86 87 Vessel 5 99 99 6889 94 Vessel 6 104 99 90 82 89 Average 93 100 87 88 92 RSD 13.1 3.8 11.84.3 5.5

Dry blend 3 mg lot 1528-2907-RD 500-mL 15 min 30 min 45 min 60 minVessel 1 91 96 97 96 Vessel 2 96 95 97 96 Vessel 3 96 97 97 97 Vessel 495 102 100 100 Vessel 5 97 96 96 97 Vessel 6 92 99 98 98 Average 94 9798 97 RSD 2.7 2.5 1.1 1.4

Dry blend 1 mg lot 1528-2906-RD 150-mL 15 min 30 min 45 min 60 minVessel 1 65 92 96 99 Vessel 2 49 91 95 96 Vessel 3 46 88 96 97 Vessel 444 96 101 102 Vessel 5 39 78 93 99 Vessel 6 57 90 95 96 Average 50 89 9698 RSD 18.8 7 2.8 2.4

Dry blend 0.3 mg lot 1528-2890-RD 50-mL 15 min 30 min 45 min 60 minVessel 1 57 94 100 105 Vessel 2 60 96 100 105 Vessel 3 86 93 94 95Vessel 4 76 90 91 101 Vessel 5 69 90 97 106 Vessel 6 68 95 97 97 Average69 93 97 102 RSD 15.6 2.8 3.4 4.5

Capsule Dissolution at 45 minutes Lot 5 C. 25 C. 30 C. 40 C. (strength)COA 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 M 2011F101A  98%  99% 95%  95%  95%  92%  95%  94% 93%  97%  93% 90% 92% (0.3 mg) 2011F099A 96%  95%  95%  95%  91%  93%  94%  93% 90%  95%  95% 92% 93%   (1 mg)2011F100A  99% 101%  97%  97% 100%  95%  95%  98% 95%  95%  96% 93% 95%  (3 mg) 11H141 101% 102% 101% 101% 105%  96% 106% 102% 97% 103%  99%96% 98% (0.3 mg) 11H152  96%  96%  99%  97%  96%  99%  97%  96% 96%  98% 96% 96% 98%   (1 mg) 11H140 102% 102% 102% 101% 105% 100%  97% 102% 99%102% 101% 99% 96%   (3 mg)

Dissolution 75-Rpm:

The tables below show a few examples where the stirring rate wasincreased slightly to 75-rpm to give more consistent results andindicates stable dissolution after accelerated storage of 1 or 2 monthsat 40 C 75% relative humidity.

Dry blend 0.3 mg lot 1528-2850-RD 1M 40 C./75RH 75-rpm 50-mL 15 min 30min 45 min 60 min Vessel 1 75 80 80 81 Vessel 2 61 75 80 82 Vessel 3 6581 83 84 Vessel 4 78 86 84 85 Vessel 5 66 79 83 84 Vessel 6 62 79 84 86Average 68 80 82 84 RSD 10.3 4.5 2.3 2.2

Dry blend 1 mg lot 1528-2906A-RD 2M 40 C./75RH 75-rpm 50-mL 15 min 30min 45 min 60 min Vessel 1 69 84 88 88 Vessel 2 62 82 84 85 Vessel 3 6582 85 85 Vessel 4 58 70 80 79 Vessel 5 59 77 82 81 Vessel 6 68 80 83 84Average 64 79 84 84 RSD 7.2 6.4 3.3 3.8

2855-RD Dissolution:

The tables below are all the dissolution profiles of batch 1528-2850-RDand indicate stable drug release over time.

Initial Percent Dissolved Vessel 15 30 45 60 1 84% 99% 104%  104%  2 28%80% 89% 92% 3 68% 83% 91% 95% 4 56% 79% 88% 98% 5 29% 83% 94% 98% 6 74%85% 93% 96% Mean 57% 85% 93% 97% RSD 41.20%   8.50%   6.00%   4.20%  

1 M 40 C./75 RH 2 M 30 C./65 RH 3 M 30 C./65 RH 3 M 25 C./60 RH OxyGuardPackaging OxyGuard OxyGuard OxyGuard 15 30 45 60 15 30 45 60 15 30 45 6015 30 45 60 Vessel min min min min min min min min min min min min minmin min min 1 35 74 88 93 47 67 80 90 76 83 87 88 44 62 78 85 2 46 74 7985 57 80 91 95 65 79 86 91 70 89 94 97 3 39 78 84 88 43 55 63 71 64 8492 97 48 62 74 79 4 59 82 92 94 753 92 98 101 71 85 90 94 65 92 98 103 522 82 89 92 38 64 81 92 60 75 81 87 72 86 93 96 6 4 20 44 61 54 94 99101 55 74 81 87 53 74 78 84 Average 34 68 79 86 52 75 85 92 65 80 86 9159 78 86 91 RSD 57 35 23 14 25 21 16 12 11.7 5.7 5.3 4.6 20.1 17.4 12.110.4 1 M 40 C./75 RH 2 M 30 C./65 RH 3 M 30 C./65 RH 3 M 25 C./60 RHHDPE Bottle HDPE HDPE HDPE 15 30 45 60 15 30 45 60 15 30 45 60 15 30 4560 Vessel min min min min min min min min min min min min min min minmin 1 61 78 85 89 78 97 100 103 58 72 79 85 54 70 83 92 2 63 83 90 92 7793 97 98 51 72 83 90 66 81 88 92 3 66 79 84 91 41 59 71 78 53 84 91 9410 29 50 66 4 25 44 64 77 50 65 73 78 66 89 94 95 69 81 88 92 5 47 67 7580 37 59 72 83 48 66 75 81 68 83 92 97 6 57 71 80 85 6 21 39 52 85 94 9699 82 91 94 97 Average 53 70 80 86 48 66 75 82 60 80 86 91 58 73 83 89RSD 28 20 12 7 56 42 29 22 22.6 14 9.7 7.3 43 30.6 19.6 13.3 1 M 40C./75 RH 2 M 30 C./65 RH 3 M 30 C./65 RH 3 M 25 C./60 RH BlisterPackaging Blister Blister Blister 15 30 45 60 15 30 45 60 15 30 45 60 1530 45 60 Vessel min min min min min min min min min min min min min minmin min 1 36 69 85 90 61 91 98 100 82 95 100 102 53 71 81 90 2 41 69 8488 57 82 94 100 31 48 63 74 27 57 80 87 3 67 96 97 98 63 87 96 100 69 7782 85 70 78 87 92 4 54 83 94 104 36 80 96 100 29 41 55 69 52 66 74 87 510 46 64 79 45 61 75 83 84 94 95 97 25 48 66 80 6 70 91 96 100 87 100102 104 74 84 89 82 50 74 82 84 Average 47 76 87 93 58 83 93 98 62 73 8185 46 66 78 87 RSD 48 25 14 10 30 16 10 8 40.5 32.1 22.4 14.9 37.0 17.09.2 5.3

Bathes 2850-RD, 2850B-RD, 2851-RD, and 500-55 were also tested in thesimilar fashion and all showed stable drug release over time.

We claim:
 1. A method for treating chronic constipation in a humansubject comprising orally administering to said human subject acomposition consisting of SEQ ID NO:1 wherein the peptide is a [4,12;7,15] bicycle, an inert low moisture carrier, and a lubricant, andwherein the peptide has a chromatographic purity of no less than 91%after storage for at least three months.
 2. The method of claim 1,wherein the constipation is associated with irritable bowel syndrome orchronic idiopathic constipation.
 3. A method of treating or alleviatinga symptom associated with chronic idiopathic constipation or irritablebowel syndrome in a human subject comprising orally administering tosaid human subject a composition consisting of SEQ ID NO:1 wherein thepeptide is a [4,12; 7,15] bicycle, an inert low moisture carrier, and alubricant, and wherein the peptide has a chromatographic purity of noless than 91% after storage for at least three months.
 4. The method ofclaim 3, wherein the symptom is constipation or abdominal pain.
 5. Themethod of claim 1, further comprising administering to said patient aneffective dose of an inhibitor of cGMP-dependent phosphodiesteraseeither concurrently or sequentially with said guanylate cyclase receptoragonist.
 6. The method of claim 5, wherein said inhibitor ofcGMP-dependent phosphodiesterase is selected from the group consistingof sulindac sulfone, zaprinast, and motapizone.
 7. The method of claim1, further comprising administering to said patient an effective dose ofa laxative.
 8. The method of claim 3, further comprising administeringto said patient an effective dose of an inhibitor of cGMP-dependentphosphodiesterase either concurrently or sequentially with saidguanylate cyclase receptor agonist.
 9. The method of claim 8, whereinsaid inhibitor of cGMP-dependent phosphodiesterase is selected from thegroup consisting of sulindac sulfone, zaprinast, and motapizone.
 10. Themethod of claim 3, further comprising administering to said patient aneffective dose of a laxative.
 11. The method of claim 1, wherein theinert low moisture carrier is microcrystalline cellulose.
 12. The methodof claim 1, wherein the lubricant is magnesium stearate.
 13. The methodof claim 1, wherein the inert low moisture carrier is microcrystallinecellulose and the lubricant is magnesium stearate.
 14. The method ofclaim 3, wherein the inert low moisture carrier is microcrystallinecellulose.
 15. The method of claim 3, wherein the lubricant is magnesiumstearate.
 16. The method of claim 3, wherein the inert low moisturecarrier is microcrystalline cellulose and the lubricant is magnesiumstearate.